CA2192797C - Indolinone compounds for the treatment of disease - Google Patents

Indolinone compounds for the treatment of disease Download PDF

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CA2192797C
CA2192797C CA002192797A CA2192797A CA2192797C CA 2192797 C CA2192797 C CA 2192797C CA 002192797 A CA002192797 A CA 002192797A CA 2192797 A CA2192797 A CA 2192797A CA 2192797 C CA2192797 C CA 2192797C
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indolinone
methylene
alkyl
aryl
cells
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CA2192797A1 (en
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Peng Cho Tang
Li Sun
Gerald Mcmahon
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Sugen LLC
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Abstract

The present invention relates to organic molecules capable of modulating tyrosine kinase signal transduction in order to regulate, modulate and/or inhibit abnormal cell proliferation.

Description

INI)OhINONE COMPOUNDS FOR THE TREATMENT OF DISEASE
1. INTRODUCTI IJN
The present invention relates to novel compounds capable of modulating, :regulating and/or inhibiting tyrosine kinase signal transduction. The present invention is also directed to methods of regulating, modulating or inhibiting tyrosine kinases, whether of the receptor or non-receptor class, for the prevention and/or treatment of disorders related to unregulated tyrosine kinase signal transduction, including cell proliferat:ive and metabolic disorders.
2. BACKGROUND OF THE INDENTION
Protein tyrosine kinases (PTKs) comprise a large and diverse class o:E proteins having enzymatic activity. The PTKs play an im~~ortant role in the control of cell growth and differentiation (for review, see Schlessinger & Ullrich, 1992, Neuron 9::383-391).
For example=, receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signalling molecules that facilitate the appropriate cellular response (e.g., cell division, .
metabolic effects to the extracellular microenvironment).
See, Schlessing~=r and LTllrich, 1992, Neuron 9:383-391.
With respe~~t to receptor tyrosine kinases, it has been shown also that tyrosine phosphorylation sites function as high-affinity binding sites for SH2 (src homology) domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423;
Songyang e~ al., 1994, :~~01. Cell. Biol. 14:2777-2785);

A

l .?
L I ~J~I ~~
Songyang et a.Z., 1993, Cell 72:767-778; and Koch et al., 1991, Science 252:668-678. Several intracellular substrate proteins that associate with receptor tyrosine kinases (RTKs) have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and associate with catalytically active molecules. Songyang et al., 1993, Ce_L1 72:767-778. The specificity of the interactions between receptors or proteins and SH2 domains of their substrai~es is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue.
Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed diffE:rences in their substrate phosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. These observations :suggest that the function of each receptor tyrosine kinase is .determined not only by its pattern of expression anti liga:nd availability but also by the array of downstream signal t:ransduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways :recruited by specific growth factor receptors, as well .as differentiation factor receptors.
Aberrant expression or mutations in the PTKs have been shown to lead to either uncontrolled cell proliferation (e. g.
malignant tumor growth) or to defects in key developmental processes. Consequently, the biomedical community has expended significant resources to discover the specific biological ro7_e of members of the PTK family, their function in differentiation processes, their involvement in tumorigenesis and in other diseases, the biochemical mechanisms underlying their signal transduction pathways activated upon ligand stimulation and the development of novel drugs.

Tyrosine kinases can be of the receptor-type (having extracellular, transmembrane and intracellular domains) or the non-receptor type (being wholly intracellular).
Receptor Tyrosine Kinases. The RTKs comprise a large family of transmembrane receptors with diverse biological activities. The intrinsic function of RTKs is activated upon ligand binding, which results in phosphorylation of the receptor and multiple cellular substrates, and subsequently in a variety of cellular responses. Ullrich & Schlessinger, 1990, Cell 61:203-212.
At present, at least nineteen (19) distinct RTK
subfamilies have been identified. One RTK subfamily, designated the HER subfamily, is believed to be comprised of EGFR, HER2, HER3 and HER4. Ligands to the HER subfamily of receptors include epithelial growth factor (EGF), TGF-a, amphiregulin, HB-EGF, betacellulin and heregulin.
A second family of RTKs, designated the insulin subfamily, is comprised of the INS-R, the IGF-1R and the IR-R. A third family, the "PDGF" subfamily includes the PDGF a and ~i receptors; CSFIR, c-kit and FLK-II. Another subfamily of RTKs, identified as the FLK family, is believed to be comprised of the Kinase insert Domain-Receptor fetal liver kinase-1 (KDR/FLK-1), the fetal liver kinase 4 (FLK-4) and the fms-like tyrosine kinase 1 (flt-1). Each of these receptors was initially believed to be receptors for hematopoietic growth factors. Two other subfamilies of RTKs have been designated as the FGF receptor family (FGFRl, FGFR2, FGFR3 and FGFR4) and the Met subfamily (c-met and Ron) .
Because of the similarities between the PDGF and FLK
subfamilies, the two subfamilies are often considered together. The known RTK subfamilies are identified in Plowman et al., 1994, DN&P 7(6):334-339, The Non-Receptor Tyrosine Itiaases. The non-receptor tyrosine kinases represent a collection of cellular enzymes which lack extracellular and transmembrane sequences. At present, over twenty-four individual non-receptor tyrosine kinases, comprising eleven (11) subfamilies (Src, Frk, Htk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack and LIMK) have been identified. At present, the Src subfamily of non-receptor tyrosine kinases is comprised of the largest number of PTKs and include Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
The Src subfamily of enzymes has been linked to oncogenesis.
A more detailed discussion of non-receptor tyrosine kinases is provided in Bolen, 1993, Oncogene 8:2025-2031.
Many of the tyrosine kinases, whether an RTK or non-receptor tyrosine kinase, have been found to be involved in cellular signaling pathways leading to cellular signal assays signalling pathways leading to pathogenic conditions, including cancer, psoriasis and hyper immune response.
Development Of Compounds To Modulate The PTIts. In view of the surmised importance of PTKs to the control, regulation and modulation of cell proliferation and the diseases and disorders associated with abnormal cell proliferation, many attempts have been made to identify receptor and non-receptor tyrosine kinase "inhibitors" using a variety of approaches, including the use of mutant ligands (U.S. Application No.
4,966,849), soluble receptors and antibodies (Application No.
W0 94/10202; Kendall & Thomas, 1994, Proc. Nat'1 Acad. Sci 90:10705-09; Kim, et al., 1993, Nature 362:841-844), RNA
ligands (Jellinek, et al., Eiochemistry 33:10450-56);
Takano, et al., 1993, Mol. Hio. Cell 4:358A; Kinsella, et al., 1992, Exp. Cell Res. 199:56-62; Wright, et al., 1992, J.
Cellular Phys. 152:448-57) and tyrosine kinase inhibitors (WO
94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Patent No. 5,330,992; Mariani, et al., 1994, Proc. Am. Assoc. Cancer Res. 35:2268).
More recently, attempts have been made to identify small molecules which act as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridyl-quinolones .. ' ~ ~ ~~i 97 (U.S. Patent :~o. 5,330,992) have been described generally as tyrosine kina;se inhibitors. Styryl compounds (U. S. Patent No. 5,217,999), styryl-substituted pyridyl compounds (U. S.
Patent No. 5,:302,606), certain quinazoline derivatives (EP
Application No. 0 566 266 A1), seleoindoles and selenides (PCT WO 94/03~~27), tricyclic polyhydroxylic compounds (PCT WO
92/21660) and benzylphosphonic acid compounds (PCT WO
91/15495) havc: been described as compounds for use as tyrosine kina:~e inhibitors for use in the treatment of 1o cancer.
The ideni~ification of effective small compounds which specifically _Lnhibit signal transduction by modulating the activity of receptor and non-receptor tyrosine kinases to regulate and modulate abnormal or inappropriate cell proliferation is therefore desirable and the object of this invention.
3. SUMMARY OF THE INVENTION
The present invention relates to organic molecules capable of modulating, regulating and/or inhibiting tyrosine kinase signal transduction. Such compounds are useful for the treatment of di:~eases related to unregulated TKS
transduction, including cell proliferative diseases such as cancer, atherosclerosis, arthritis and restenosis and metabolic disE:ases :such as diabetes.
In one illustrative embodiment, the compounds of the present invention have the formula:
OR

X u (I) / _Rs R5 w /~ R2 'N
R~ R~
and pharmaceutically acceptable salts thereof, wherein ~i92797 R1 is H c>r alkyl;
RZ i s O c>r S ;
R3 is hydrogen;
R4, R5, R", and R, are each independently selected from the group con;~isting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, S02NRR' , S03R, SR, NO2, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CHz ) nC02R, and CONRIZ' ;
R3, , R5, , .and R6" are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOzNRR', S03R, SR, NO2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, ( CHz ) nCO2R, and CONRR' ;
n is 0-3;
X is Br, C1, F or I;
R is H, alkyl or aryl; and R' is H, alkyl or aryl.
In another illustrative embodiment, the compounds of the 2o present invention have the formula:
NRaRb Rs v I
RS' (II) .
~R' R. ~.~ s Rs ~ 'N
R~ R~
and pharmaceutically acceptable salts thereof, wherein R1 is H or alky7L;
RZ is O or S;
R3 is hydrogen;
R4, R,, R6, and R, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, all~aryloxy, halogen, trihalomethyl, S(O)R, SOZNRR' , S03R, SR, N02, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CH2 ) nCOaR, and CONRR' ;
Rz. , R3, , R5. , and R6, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, S02NRR' , S03R, SR, NOZ, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CHZ ) nCOaR, and CONRR' ;
Ra and Rb are each independently selected from the group consisting of H, alkyl and C(O)R, or NRaRb taken together may be a heterocyclic ring of from 3 to 8 atoms optionally substituted at one or more positions with hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, S02NRR', S03R, SR, NOZ, NRR', OH, CN, C (O) R, OC (O) R, NHC (O) R, (CHz) "C02R, or CONRR' ;
n is 0-3;
R is H, alkyl or aryl; and R' is H, alkyl or aryl.
In yet another illustrative embodiment, the compounds of the present invention have the formula:
A

Rs (III) ~~~'N
s 3o R~ R~
and pharmaceutically acceptable salts thereof, wherein R1 is H or alkyl;
RZ is O or S;
35 R3 is hydrogen;
R4, R5, R6, and R7 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, S02NRR', S03R, SR, N02, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, ( CHZ ) nCO2R, and CONRR' ;
A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, and tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S (O) R, SO~NRR' , S03R, SR, NOZ, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, (CHZ) nCO2R Or CONRR' ;
n is 0-3;
R is H, alkyl or aryl; and R' is H, alkyl or aryl.
In still another illustrative embodiment, the compounds of the present invention have the formula:
2 5 ~ ~ R5 RO
( Iv ) Rs R4 CRs R5 ~
~ /'-R2 R~ ~ ' N
3 0 R~ R~
and pharmaceutically acceptable salts thereof, wherein:
R1 is H or alkyl;
RZ is O or S;
35 R3 is hydrogen;
R4, R5, R6, and R, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, - g _ aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, S02NRR' , S03R, SR, NOa, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CH2 ) nCOZR, and CONRR' ;
R3. , R4. , R5. , and R6, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOZNRR' , S03R, SR, NO2, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CHz ) nCO2R, and CONRR' ;
n is 0-3;
R is H, alkyl or aryl; and R' is H, alkyl or aryl.
In a final illustrative embodiment, the compounds of the.
present invention have the formula:
~5 (V) R

Re and pharmaceutically acceptable salts thereof, wherein:
Rl is H or alkyl;
Rz is O or S;
R3 is hydrogen;
R4, R5, Rs, and R~ are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOZNRR' , S03R, SR, NOz, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CH2 ) nCO2R and CONRR' ;
RZ. , R3. , R5. , and R6, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, g R~ rct ~ ~ '~~ 797 S02NRR' , S03R, SR, NO2, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CHZ ) nCO2R and CONRR' ;
n is 0-3;
Z is Br, C1, F,, I, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-x~utyl or tert-butyl;
R is H, alkyl or aryl; and R' is H, alkyl or aryl.
The present invention is further directed to pharmaceutical compositions comprising a pharmaceutically effective amount of the above-described compounds of formulae I-V and a pharmaceui:ically acceptable carrier or excipient.
Such a composition is believed to modulate signal transduction ~~y a tyrosine kinase, either by inhibition of catalytic activity, affinity to ATP or ability to interact with a substrate.
More particularly, the compositions of the present invention may be included in methods for treating diseases comprising proliferation, fibrotic or metabolic disorders, for example cancer, fibrosis, psoriasis, atherosclerosis, arthritis, and. other- disorders related to abnormal vasculogenesis and/or angiogenesis, such as diabetic retinopathy.
4. DETAILED DESCR7CPTION OF THE INVENTION
4.1. Definitions "Pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic: acid, p-toluenesulfonic acid, salicylic acid and the like.
"Alkyl" refers to a straight-chain, branched or cyclic saturated aliphatic hydrocarbon. Preferably, the alkyl group has 1 to 12 carbons.. More preferably, it is a lower alkyl of from 1 to 7 carbons,, more preferably 1 to 4 carbons. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like. The alkyl group may be optionally substituted with one or more substituents se7.ected from the group consisting of hydroxyl, cyano, alkoxy, =O, =S, N02, halogen, N(CH3)2 amino, and SH.
"Alkenyl" :refers to a straight-chain, branched or cyclic unsaturated hydrocarbon group containing at least one carbon-carbon double bend. Preferably, the alkenyl group has 1 to 12 carbons. More pre:ferably~it is a lower alkenyl of from 1 to 7 carbons, mere preferably 1 to 4 carbons. The alkenyl group may be optionally substituted with one or more substituents selectedl from the group consisting of hydroxyl, cyano, alkoxy, --O, _~~, NOz, halogen, N(CH3)2 amino, and SH.
"Alkynyl" refer; to a straight-chain, branched or cyclic unsaturated hydrocarbon containing at least one carbon-carbon triple bond. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkoxy, =O, =S, N02, halogen, N(CH3)z amino, and SH.
"Alkoxy" refers to an "-Oalkyl" group.
"Aryl" refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, het:erocyclic aryl and biaryl groups. The aryl group may be optionally substituted with one or more substituents selected from the group consisting of halogen, trihalomethyl, hydroxyl, SH, OH, NO2, amine, thioether, cyano, alkoxy, alkyl, and amino.
"Alkaryl" refer: to an alkyl that is covalently joined to an aryl group. Preferably, the alkyl is a lower alkyl.
"Carbocyclic aryl" refers to an aryl group wherein the ring atoms are carbon.
"Heterocyc:lic aryl" refers to an aryl group having from 1 to 3 heteroat:oms a:~ ring atoms, the remainder of the ring atoms being carbon. Heteroatoms include oxygen, sulfur, and A

nitrogen. Thus, hets:rocyclic aryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imid.azoly:l and the like.
"Amide" refers 1:o -C (O) -NH-R, where R is alkyl, aryl, alkylaryl or hydrogen.
"Thioamide:" refEars to -C(S)-NH-R, where R is alkyl, aryl, alkylaryl. or h~tdxogen.
"Amine" re:f ers 1.o a -N ( R' ) R' ' group, where R' and R' ' are independently se:Lected from the group consisting of alkyl, aryl, and alk~~laryl. ' "Thioether" refers to -S-R, where R is alkyl, aryl, or alkylaryl.
"Sulfonyl" refers to -S (O) 2-R, where R is aryl, C (CN) =C-aryl, CHZCN, al:kyaryl., sulfonamide, NH-alkyl, NH-alkylaryl, or NH-aryl.
4.2. The ~:nvent:ion The present invention relates to compounds capable of regulating and/or modulating tyrosine kinase signal transduction and more particularly receptor and non-receptor tyrosine kinase: signal transduction.
Receptor tyrosine kinase mediated signal transduction is initiated by a}arace:llular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular ~~ignal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signalling mols:cules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects to the extracellular microe:nvironment). See, Schlessinger and Ullrich, 1992, Neuron 9:383-391.
It has bes:n shown that tyrosine phosphorylation sites in growth factor receptors function as high-affinity binding sites for SH2 ~;src homology) domains of signaling molecules.
Fantl et al., 7_992, ~~e11 69:413-423; Songyang et al., 1994, Mol. Cell. Bio~. 14:2777-2785); Songyang et al., 1993, Cell A

72:767-778; and Koch et al., 1991, Science 252:668-678.
Several intracellular substrate proteins that associate with receptor tyrosine kinases have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and associate with catalytically active molecules. Songyang et al., 1993, Cell 72:767-778. The specificity of the interactions between receptors and SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed differences in their substrate phosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. These observations suggest that the function of each receptor tyrosine kinase is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors.
Tyrosine kinasE: signal transduction results in, among other responses, ce7~~1 proliferation, differentiation and metabolism. Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, leukemia, glioblastoma, hemangioma, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy (or other disorders related to uncontrolled angiogE.nesis and/or vasculogenesis).
This invention is therefore directed to compounds which regulate, modulate and/or inhibit tyrosine kinase signal transduction x~y affE:cting the enzymatic activity of the RTKs and/or the non.-receptor tyrosine kinases and interfering with the signal tra.nsducE:d such proteins. More particularly, the 2192797 ~e present invention is directed to compounds which regulate, modulate and/or: inhibit the RTK and/or non-receptor tyrosine kinase mediated signal transduction pathways as a therapeutic approach to cure leukemia and many kinds of solid tumors, including but not limited to carcinoma, sarcoma, erythroblastoma, g:lioblastoma, meningioma, astrocytoma, melanoma and myoblas;toma. Indications may include, but are not limited t:o brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers, blood cancers, lung ~~ancers and bone cancers.
4.3. The c:ompou:nds In one embodiment, the invention provides compounds having the formula:
R' R' s (I) X

~ / R2 Rg ~ ~N
R~ R~
and the pharmaceutically acceptable salts thereof, wherein R1 is H or alkyl.;
R2 is O or S;
R3 is hydrogen;
R4, R5, R5, and Ft, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOzNRR', S03R, SR, NO,, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, ( CH2 ) nCO2R, and CONRR' ;
R3, , RS. , and R5, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO,NRR', SO~R, SR, N02, rIRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CH, ) nCOZR, and CONRR' ;

A

z j ~~z~ ~~
n is 0-3;
X is Br, C1, F or I; and R is H, ~~lkyl or aryl; and R' is H, alkyl or aryl.
In a preferred embodiment of the compounds of formula I, R3' is selected from the group consisting of methyl, ethyl, n-propyl, iso-p:ropyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl; and RS' is selected to from the grou~g consisting of hydrogen, methyl, ethyl, n-propyl, iso-p:ropyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl.
A particularly preferred compound of formula I is 3-(2-chloro-4-hydr~~xybenzylidenyl)-2-indolinone.
In another embodiment, the invention provides compounds having the formula:
NRaRb 2 0 ~ ~ Rs (II) R2 Rs W
~R2 Rs ~ ' N
R~ R~
and the pharm;~ceutically acceptable salts thereof, wherein R1 is H or alkyl;
RZ is O or S;
R3 is hydrogen;
R4, R5, R6, and R., are each independently selected from the group con,~isting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alka:ryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOzNRR' , S03R, SR, Nf02, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, (CHz) nCOzR, and CONR:R' ;
R2, , R3, , R5, , and R6, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, S02NRR' , S03R, SR, N02, NRR' , OH, CN, , C (O) R, OC (O) R, NHC (O) R, ( CHz ) nCO2R, and CONRR' ;
Ra and Rb are each independently selected from the group consisting of H, alkyl and C(O)R, or NRaRb taken together may be a heterocyclic ring of from 3 to 8 atoms'optionally substituted at one or more positions with hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOZNRR',.S03R, SR, N02, NRR', OH, CN, C (O) R, OC (O) R, NHC (O) R, (CHz) nC02R, or CONRR' ;
n is 0-3;
R is H, alkyl or aryl; and R' is H, alkyl or aryl.
In a preferred embodiment of the compounds of formula II, R3' is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl; and RS' is selected from the group consisting of hydrogen, methyl, ethyly n-propyl, iso-propyl, n-butyl, iso-butyl, tent-butyl, halogen, aryl and OR, where R is H_, alkyl or aryl.
A particularly preferred compound of.formula II is 3-(4-Dimethylaminobenzylidenyl)-2-indolinone.
In yet another embodiment, the invention provides compounds having the formula:

(III) Rs and the pharmaceutically acceptable salts thereof, wherein R1 is H or alkyl;
RZ is O or S;

R~ is hydrogen;
R" RS, Rs, and R, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, S02NRR' , S03R, SR, NOZ, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CH= ) nCOaR, and CONRR' ;
A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, l0 isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole,. 1,3,4-thiadiazole, 1,2,3,4-thiatriazole , 1,2,3,5-thiatriazole, and tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy; alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOzNRR', S03R, SR, NOz, NRR', OH, CN, c (o) R, oc (o) R, NHC (o) R; (cHz) "C02R or coNRR' ;
n is 0-3;
ao R is H, alkyl or aryl; and R' is H, alkyl or aryl.
In a preferred embodiment of the invention, the compound of formula III is 3-[(2,4-Dimethylpyrrol-5-yl)methylene]-2-indolinone.
In still another embodiment, the invention provides compounds having the formula:
(IV) and the pharmaceutically ~5r acceptable salts thereof, wherein:
R1 is H or alkyl;
,R2 is o or S;
R3 is hydrogen; .., Rs . R6 . and R7 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOZNRR', S03R, SR, N02, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, (CH2) nCOzR, and CONRR';
R3: , R4. , R5. , and Rs, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOZNRR' , S03R, SR, N02, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CHz ) nCOzR, and CONRR' ;
n is 0-3;
R is H, alkyl or aryl; and R' is H, alkyl or aryl.
In a preferred embodiment of the compound of formula IV, R3' is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl; and RS' is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl.
A particularly preferred compound of formula IV is 3-(2-Ethoxybenzylidenyl]-2-indolinone.
In a final embodiment, the invention provides compounds having the formula: , - 18 . -2 ~ ~~2797 (v) z R' R4 CR3 s and the pharmaceutically R ~ N
s acceptable salts R~ R1 thereof, wherein:
l0 R1 is H or alkyl;
RZ is O or S;
R3 is hydrogen;
R4 , RS , R,; , and R., are each independently selected from the group con:~isting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen; trihalomethyl, S(O)R, SOzNRR', S03R, SR, NO2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, ( CHZ ) nCOzR and CONRR' ;
R2, , R3, , R5, , and R6, are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SOZNRR' , S03R, SR, NO2, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, ( CH2 ) nCO2R and CONRR' ;
n is 0-3;
Z is Br, C1, F, I, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl;
R is H, ~~lkyl or aryl; and R' is H, alkyl or aryl.
In a preferred embodiment of the compounds of formula V, R3' is selected from the group consisting of methyl, ethyl, n-3o propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl; and RS' is selected from the grouch consisting of hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl.
A particularly preferred compound of formula V is 3-(4-Bromobenzylidenyl)-?.-indolinone.

21 ~2~~i The chemical formulae referred herein may exhibit the phenomena of tautomerism or structural isomerism. For example, the compounds described herein may be adopt a cis or trans conformation about the double bond connecting the indolinone 3-substi.tuent to the indolinone ring, or may be mixtures of cis and trans isomers. As the formulae drawing within this specification can only represent one possible tautomeric or strucaural isomeric form, it should be understood that the: invention encompasses any tautomeric or structural isomeric'. form, or mixtures thereof, which possesses the ability to regulate, inhibit and/or modulate tyrosine kinase signal transduction or cell proliferation and is not limited to any one tautomeric or structural isomeric form utilized within the formulae drawing.
In addition to the above-described compounds and their pharmaceutica:Lly acceptable salts, the invention is further directed, wheeze applicable, to solvated as well as unsolvated forms of the compounds (e.g. hydrated forms) having the ability to regulate and/or modulate cell proliferation.
The compounds described herein may be prepared by any process known to be applicable to the preparation of chemically-re_Lated compounds. Suitable processes are illustrated in the examples. Necessary starting materials may be obtainE~d by atandard procedures of organic chemistry.
An individual ~~ompound's relevant activity and efficacy as an agent to affect receptor tyrosine kinase mediated signal transduction may be determined using available techniques. F~referentially, a compound is subjected to a series of scrE:ens to determine the compound's ability to modulate, regulate and/or inhibit cell proliferation. These screens, in the order in which they are conducted, include biochemical a~;says, cell growth assays and in vivo experiments.

2192797 ~:
4 . 4 . Indicrations The compounds described herein are useful for treating disorders related to unregulated tyrosine kinase signal transduction, :Lncluding cell proliferative disorders, fibrotic disorders and metabolic disorders.
Cell prol:iferative disorders which can be treated or further studied by the present invention include cancers, blood vessel proliferative disorders and mesangial cell proliferative disorders.
Blood vesael proliferative disorders refer to angiogenic and vasculogen:ic disorders generally resulting in abnormal proliferation of blood vessels. The formation and spreading of blood vessels, or vasculogenesis and angiogenesis, respectively, May important roles in a variety of physiological ;processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. They also play a pivotal role in cancer development. Other examples of blood weasel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilaa~e, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness. Conversely, disorders related to the shrinkage, contraction or closing of blood vessels, such as re~~tenosis, are also implicated.
Fibrotic disorders refer to the abnormal formation of extracellular matrix. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders.
Hepatic cirrohis is characterized by the increase in extracellular matrix; constituents resulting in the formation of a hepatic scar. Hepatic, cirrhosis can cause diseases such as cirrhosis of the liver. An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis. Lipocytes appear to play a major role in hepat=is cirrhosis. Other fibrotic disorders implicated include atherosclerosis (see, below).
Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells. Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephroypathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. The PDGF-R has been implicated in the maintenance of mesanarial cell proliferation. Floege et al., 1993, Kidney International 43:47S-54S.
PTKs have been associated with such cell proliferative disorders. For example, some members of the RTK family have been associated with the development of cancer. Some of these receptors, like: the EGFR (Tuzi et al., 1991, Br. J.
Cancer 63:227-233; Torp et al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science 244:707-712) and the PDGF-R (Kumabe et al., 1992, Oncogene 7:627-633) are overexpressed in many tumors and/or persistently activated by autocrine loops. In fact, in the most common and severe cancers these receptor overexpressions (Akbasak and Suner-Akbasak et al., 1992,, J. Neurol. Sci. 111:119-133; Dickson et al., 1992, Cancer Treatment Res. 61:249-273; Korc et al., 1992, J. CZin. Invest.. 90:1352-1360) and autocrine loops (Lee and Donoghue, 1992, ~T. Cell. Biol. 118:1057-1070; Korc et al., supra; Akh~asak and Suner-Akbasak et al., supra) have been demonstrated. For example, the EGFR receptor has been associated with. squamous cell carcinoma, astrocytoma, giioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 h.as been associated with breast, ovarian, gastric, lung, pancrc_as and bladder cancer. The PDGF-R has been associated with glioblastoma, lung, ovarian, melanoma and prostate cancer. The TRK c-met has been generally associated wits. hepat:ocarcinogenesis and thus hepatocellular carcinoma. Adc.itionally, c-met has been linked to malignant tumor formation. More specifically, the RTK c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic and gastric carcinoma, leukemia and lymphoma.
Additionally, over-expression of the c-met gene has been detected in patients with Hodgkins disease, Burkitts disease, and the lymphoma cel:~ line.

A

' ~ ~ ~~ % ~'7 The IGF-IR, in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with sevE~ral types of cancers. For example, IGF-I
has been implicated as an autocrine growth stimulator for several tumor types,, e.g. human breast cancer carcinoma cells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and small lung tumor cells (Macauley et al., 1990, Cancer Res.
50:2511-2517). In addition, IGF-I, integrally involved in the normal growth and differentiation of the nervous system, appears to be an aui=ocrine stimulator of human gliomas.
Sandberg-Nordg:vist Eat al., 1993, Cancer Res. 53:2475-2478.
The importance: of the IGF-IR and its ligands in cell proliferation is fu~_-ther supported by the fact that many cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stE:m cells of the bone marrow) are stimulated to grow by IGF-I. Goldring and Goldring, 1991, Eukaryotic Genie Expression 1:301-326. In a series of recent publications, Baserga even suggests that IGF-I-R plays a central role i.n the mechanisms of transformation and, as such, could be: a preferred target for therapeutic interventions for a broad spectrum of human malignancies.
Baserga, 1995, Cancer Res. 55:249-252; Baserga, 1994, Cell 79:927-930; C'.oppola et al., 1994, Mol. Cell. Biol. 14:4588-4595.
The association between abnormalities in RTKs and disease are not resi~ricted to cancer, however. For example, RTKs have been associated with metabolic diseases like psoriasis, diabetes mellitus, wound healing, inflammation, and neurodegenerative diseases. For example, the EGF-R is indicated in c:ornea:l and dermal wound healing. Defects in the Insulin-R and the IGF-1R are indicated in type-II
diabetes mellitus. A more complete correlation between specific RTKs and their therapeutic indications is set forth in Plowman et al., :L994, DN&P 7:334-339.
Not only receptor type tyrosine kinases, but also many cellular tyro~cine k:inases (CTKs) including src, abl, fps, ~19~i9i yes, fyn, lyn, lck, blk, hck, fgr, yrk (reviewed by Bolen et al., 1992, FASEB J. 6:3403-3409) are involved in the proliferative and meaabolic signal transduction pathway and thus in indications of the present invention. For example, mutated src (v-src) has been demonstrated as an oncoprotein (pp60"-S=°) in chicken. Moreover, its cellular homolog, the proto-oncogene pp60°-Sr° transmits oncogenic signals of many receptors. For example, overexpression of EGF-R or HER2/neu in tumors leads to t:he constitutive activation of pp60~-5r~, which is characteri~>tic for the malignant cell but absent from the normal cell. On the other hand, mice deficient for the expression of c-~src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders.
Similarly, Zap 70 i~~ implicated in T-cell signalling.
Furthermore, the identification of CTK modulating compounds to augment: or even synergize with RTK aimed blockers is an aspects of the present invention.
Finally, both RTKs and non-receptor type kinases have been connected to hyperimmune disorders.
4.5. Pharmaceutical Formulations And Routes Of Administration The compounds described herein can be administered to a human patient per se, or in pharmaceutical compositions where it is mixed with su~'~table carriers or excipient(s).
Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest Edition.
4.5.1. Routes Of Administration.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration.; parE:nteral delivery, including intramuscular, subcutaneous, intrarnedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitonea.l, ini~ranasal, or intraocular injections.

Alternats:ly, one may administer the compound in a local rather than systemic: manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific: antibody. The liposomes will be targeted to and taken up ~;elect~Lvely by the tumor.
4.5.2. Composition/Formulation.
The pharnaceut~:cal compositions of the present invention may be manufacaured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levic~ating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising exc:ipienta and auxiliaries which facilitate processing of the acaive compounds into preparations which can be used ph.armace:utically. Proper formulation is dependent upon. the route of administration chosen.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal admini~~tration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration; the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
Such carriers enable: the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations f'or oral use can be obtained by combining the active compounds with a solid excipient, optionally grinding a resulting mixture, and processing the A

X192797 ~s mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients arcs, in particular, fillers such as sugars, including laci~ose, sucrose, mannitol, or sorbitol; cellulose preparations :such as, for example, maize starch, wheat starch, rice :March, potato :starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylc~ellulose, and/or polyvinylpyrrolidone (PVP).
If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereo:E such as sodium alginate.
Dragee cares are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionall~~ contain gum arabic, talc, polyvinyl pyrrolidone, c~arbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterise different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-:Eit capsules made of gelatin, as well as soft, sealed capsulcas made of gelatin and a plasticizes, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
For bucc<31 administration,the compositions may take the form of tables=s or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to 1_he present invention are conveniently delivered in the form o:E an aerosol spray presentation from pressurized packs or a nehuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra:Eluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds :may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous ini:usion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may tal'~ce such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents :such as suspending, stabilizing and/or dispersing agE:nts.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-:soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles inclL:de fai~ty oils such as sesame oil, or synthetic fatty acid esters, :such as ethyl oleate or triglycerides, or liposomes. Ag:ueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, th.e suspension may also contain suitable stabilizers or agent, which increase the solubility of the compounds to allow i:or the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be fonaulated in rectal compositions ~;uch a;s suppositories or retention enemas, e.g., containing corwentional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.
Such long acting fo:cmulations may be administered by implantation (;for example subcutaneously or intramuscularly) or by intramu~~cular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oi7_) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-so:Lvent system. VPD is a solution of 3% w/v benzyl alcoho:L, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w,~v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:DSW) consists of V1?D diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds wel:L, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubilit~t and toxicity characteristics. Furthermore, the identity of the. co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of po:Lysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polye~thyler.~e glycol, e.g. polyvinyl pyrrolidone; and other sugars ~~r pol.ysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes'and emulsions are well known examples of delivery vehicles or A

. ~ 2192797 carriers for hydrophobic drugs. Certain organic solvents such as dimethylsul.foxide also may be employed, although usually at the cost: of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been. established and are well known by those skilled in th.e art. Sustained-release capsules may, depending on 'their chemical nature, release the compounds for a few weeks u~p to over 100 days. Depending on the chemical nature and the= biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carri~ars or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Many of l.he PTK modulating compounds of the invention may be providEad as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acei~ic, lactic, tartaric, malic, succinic, etc.
Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
4 . 5 ,. 3 . Ef f ective Dosage .
Pharmaceutical compositions suitable for use in the present invent=ion include compositions wherein the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the ca~~ability of those skilled in the art, especially in light of tree detailed disclosure provided herein.

A

___ , t~. ~ .,~~ 1 I' For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture a:~says. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the ICSO as determined in cell culture (i.e., the concentration of the test compound which achieve; a half-maximal inhibition of the PTK
activity). Such ini:ormation can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDSO (the dose lethal to 50% of the population) and the EDSO (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LDSO and EDso . Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulatingf concentrations that include the EDso with little or no toxicity. The dosage may vary within this range depending upon the dlosage form employed and the route of administration utilized. The exact formulation, route of administration and f.osage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1).
Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to 'maintain the kinase modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of the kinase using the assays described herein. Dosages necessary to a~~hieve the MEC will depend on individual characteristics and. route of administration. However, HPLC
assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen which maintains pla:~ma levels above the MEC for 10-90% of the time, preferably be~~ween 30-90% and most preferably between 50-90%.
In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to pl<~sma concentration.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the se=verity of the affliction, the manner of administration and the judgment of the prescribing physician.
4.5,.4. Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms contain~_ng the active ingredient. The pack may for example compr~_se metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions f:or administration. Compositions comprising a compound of tree invention formulated in a compatible pharmaceutica7_ carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition. Suitable conditions indicated on the label may inc~.ude treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
5. EXAMPLE: Compound Synthesis The compounds of the present invention may be synthesized according to known techniques. The following represent preferred methods for synthesizing the compounds of the claimed ir.:vention.
5.1. General Syntheses of 3-Substituted-2-Indolinone Analogs o ~a~i,a7 The following general methodologies were used to synthesize 3-:substituted-2-indolinone compounds of the invention.
5.1..1. Method A
A rE~action mixture of the proper oxindole (2-indolinone) (:L equiv.), the appropriate aldehyde (1.2 equiv.), and piperidine (0.1 equiv.) in ethanol (1 - 2 mL / 1 mmol oxindole;~ was stirred at 90°C for 3 - 5 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield the target compound.
5 .1,. 2 . Method B
Preparation of The Proper Aldehydes via Vilsmeier Reaction. To a solution of N,N-dimethylformamide (1.2 equiv.) in 1,2-dichloroethane (2.0 mL / 1.0 mmole of starting material) was added dropwise phosphorus oxychloride (1.2 equiv.) at 0°C. The ice-bath was removed and the reaction mixture was further stirred for 30 min. The proper starting material (1.0 equiv.) was added to the above solution portionwise and the reaction mixture was stirred at 50-70°C
for 5 h - 2 days. 'L'he reaction mixture was poured into ice-cold 1N sodium hydroxide solution (pH = 9 after mixing) and the resulting mixture was stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer was extracted with ethy:L acetate. The combined organic layer was washed with brine until pH = 7, dried over anhydrous sodium sulfate and evaporai~ed. The residue was chromatographed on a silica gel column a:Luting with a solvent mixture of ethyl acetate and hexane 1.o afford the title compound.
Synthesis for 3-Substituted-2-Indolinone Analogs.
A reaction mi~saure of the proper oxindole (2-indolinone) (1 equiv.), the appropriate aldehyde (1.2 equiv.), and piperidine (0.1 equ_w.) in ethanol (1 - 2 mL / 1 mmol oxindole) was stirred at 90°C for 3 - 5 h. After cooling, the precipitate was filtered, washed with cold ethanol and dried to yield. the target compound.

5.2. Synthesis C)f 3-Henzylidene-2-Indolinone The p~referz-ed method for synthesizing 3-benzylidene-2-indolinone is as follows: Added 123.2 ~C1 of benzaldehyde anal 40 Ecl of piperidine to a solution of 137.0 mg of oxindole in 2.0 ml methanol. Reflux the reaction mixtured for 3 hours and cool down the mixture in an ice-water bath. Filter t:he resulting precipitate, wash with cold methanol and dry in an oven at 40°C overnight. Approximately 129.0 mg of the compound was obtained using such protocol.
5.3. Synthesis 0f 3-((Pyrid-4-yl) methylene]-~2-indolinone The preferred method for synthesizing 3-[(Pyrid-4-y1 ) methylene ] -2-indo7.inone is as follows : Add 117 . 0 ~cl of 4-pyridinecarboxaldehyde and 40 p,1 of piperidine to a solution of 138.0 mg of oxindole in 2.0 ml methanol. The reaction mixture was refluxed for 3 hours and cooled down in an ice-water bath. The resulting precipitate was filtered, washed with cold methanol and dried in an oven at 40°C overnight to give 134.5 mg of the compound.
5.4. Synthesis c>f 3-[4-(morpholin-4-yl)benzylidenyl]-2-indolinone (Method H):
4-(Morpholin-4-yl)benzaldehyde. To a solution of 15 mL
of N,N-dimethylformamide in 50 mL of 1,2-dichloroethane was added dropwise 10 mL of phosphorus oxychloride at 0°C. The ice-bath was removed and the reaction mixture was further stirred for 30 min. 4-Phenylmorpholine (16.3 g) was added to the above solution portionwise and the reaction mixture was refluxed for 2 days. Triethylamine (2.5 mL) was added to the above reaction mixture and the reaction was refluxed for 2 days. The reaction mixture was poured into ice-cold 1N sodium hydroxide solution (pH = 9 after mixing) and the resulting mixture was stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer was extracted with 2 x 20 mL of dichloromethane. The combined organic layer was A

21 ~~i 9i washed with brine until pH = 7, dried over anhydrous sodium sulfate and evaporai~ed. The residue was separated on a silica gel co7.umn e:Luting with a solvent mixture of ethyl acetate and hexane i~o afford 12.95 g (68%) of the title compound as a white solid.
3-[4-(Mo=vpholin-4-yl)benzylidenyl]-2-indolinone. A
reaction mixture of 6.66 g of oxindole, 11.50 g of the 4-(morpholine-4--yl)benzaldehyde, and 5 mL of piperidine in 50 mL of ethanol was ;stirred at 90°C for 5 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 15.0 g (98%) of the title compound as a yellow solid.
5.5. Synthesis of 3-[4-(4-Formylpiperazin-1-Y1)x~enzyl:Ldenyl]-2-indolinone (Method B):
4-(4-Fora~ylpipearazin-1-yl)benzaldehyde. To a solution of 3.9 mL (30 mmole:~) of N,N-dimethylformamide in 20 mL of 1,2-dichloroet:hane was added dropwise 3.0 mL (3.9 mmoles) of phosphorus oxychloride at 0°C. The ice-bath was removed and the reaction mixtures was further stirred for 15 min. 1-Phenylpiperazine (16.0 g, 10 mmoles) was added to the above solution portionwisE~ and the reaction mixture was stirred at 50°C for 1 h. The reaction mixture was poured into ice-cold 1N sodium hydroxide solution and stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer was extracted with 2 x 20 mL of ethyl acetate. The combined organic layer was washed with brine until pH = 7, dried over anriydrou:~ sodium sulfate and evaporated. The residue was sE:paratE:d on a silica gel column eluting with a mixture of ethyl acetate and hexane to afford 9.0 g (41%) of the title compound a light yellow solid.
3-[4-(4-F'ormylpiperazin-1-yl)benzylidenyl]-2-indolinone.
A reaction mi~aure of 133.15 mg of oxindole, 228.3 mg of 4 (piperazin-1y7.)benzaldehyde, and 3 drops of piperidine in 2 mL of ethanol was si~irred at 90°C for 5 h. After cooling, the precipitate was filtered, washed with cold ethanol and 2192%;~7 dried to yield 199.5 mg (65%) of the title compound a yellow solid.
5.6. Synthesis of 3-[4-(Piperidin-1-yl)benzylidenyl]-2-indolinon~e (Method B).
4-(Piperidin-1~-yl)benzaldehyde. To a solution of 2.3 mL
(30 mmoles) of N,N-dimethylformamide in 10 mL of 1,2-dichloroethane was added dropwise 2.8 mL (30 mmoles) of phosphorus ox~~chlor:ide at 0°C. The ice-bath was removed and to the reaction mixture was stirred for 15 min. 1-Phenylpiperidine (3.2 mL, 20 mmoles) was added to the above solution portionwise and the reaction mixture was refluxed overnight. Tree reaction mixture was poured into ice-cold 2N
sodium hydroxide so:Lution and stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer was extracted with :? x 20 mL of ethyl acetate. The combined organic layer was w<~shed with brine until pH = 7, dried over anhydrous sodium su:Lfate and evaporated. The residue was separated on a~. silica gel column eluting with ethyl acetate and hexane to afford 1.5 g (400) of the title compound as a white solid.
3-[4-(Piperidin-1-yl)benzylidenyl]-2-indolinone. A
reaction mixtL:re of 134.0 mg of oxindole, 226.8 g of 4-(piperidine-1-yl)benzaldehyde, and 3 drops of piperidine in 2 mL of ethanol was :stirred at 90°C for 5 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield. 268.5 mg (88%) of the title compound as a yellow solid.
5~~~ Synt.hesis of 3-[2-Chloro-4-methoxybenzylidenyl]-2-indo~linone~.
2-Chloro-~4-methoxybenzaldehyde. The reaction mixture of 1.0 g (6.4 mmoles) of 2-chloro-4-hydroxybenzaldehyde, 4.4 g (32 mmoles) of potassium carbonate, and 1.4 g (9.6 mmoles) of methyl iodide in 10 mL of N,N-dimethylformamide was stirred at 70°C for 2 h and poured into ice water. The precipitate was filtered, washed with water, and dried at 40°C in vacuum 19>l9i oven overnight to yield 750 mg (68%) of the title compound as a light pink solid.
3-[2-Chloro-4-methoxybenzylidenyl]-2-indolinone. The reaction mixture of 487.9 mg (3.7 mmoles) of oxindole, 750 mg (4.3 mmoles) ~~f 2-chloro-4-methoxybenzaldehyde and 4 drops of piperidine in 5 mL of ethanol was heated to 90°C for 2 h and cooled to room temperature. The yellow precipitate was filtered, washed with cold ethanol, and dried at 40°C in a vacuum oven overnight to give 680.2 mg (62%) of the title compound.
5.8. Synithesis of 3-[(4-Methylthien-2-yl)methylene]-2-indolinone.
A reaction mixture of 133.0 mg of oxindole, 151.2 mg of the 4-methylthiophene-2-carboxaldehyde, and 3 drops of piperidine in 3 mL of ethanol was stirred at 90°C for 3 h.
After cooling,, the :precipitate was filtered, washed with cold ethanol, and dried to yield 147.3 mg (61%) of the title compound as a yellow solid.
5.9. Syni:hesis of 3-[(3-Methylpyrrol-2-yl)methylene]-2-indolinon~e .
A reaction mixture of 133.0 mg of oxindole, 130.9 mg of the 3-methyl~~yrrol~a-2-carboxaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90°C for 3 h.
After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 150.9 mg (67%) of the title compound as a yellow solid.
5.10. Syntlhesis of 3-[(3,4-Dimethylpyrrol-2-yl)methylene]-2-indolinone 3-[(3,4-L~imethylpyrrol-2-yl)methylene]-2-indolinone was synthesized as described in J. Heterocyclic Chem. 13:1145-1147 (1976).
Ethyl 4-methylpyrrol-3-oarboxylate. A solution of 11.86 g (0.1 moles) of ethyl crotonate and 19.50 g (0.1 moles) of p-toluenesulfanylmei~hylisocyanide in 500 mL of a 2:1 ether/dimethyl.sulfo:~ide was added dropwise into a suspension _. ~ 2192797 ~~
of 6.8 g of sodiium hydride (60% mineral oil dispension, 0.17 moles) in ether' at room temperature. Upon completion of addition the reaction mixture was stirred for 30 min and diluted with 400 mL oi: water. The aqueous layer was extracted with 3x100 mL of ether. The combined ether extracts were passed through a cohunn of alumina eluting with dichloromethane.. Ths: organic solvent was evaporated and the resulting residue was solidified on standing. The solid was washed with hexane and dried at 40°C in vacuum oven overnight to yield 12.38 g (80~>) of the title compound.
Preparation of a,4-Dimethylpyrrole. To a solution of 23 g (80 mmoles) of sodium dihydrobis(2-methoxyethoxy aluminate) was added dropwise of: a solution of 5 g (34 mmoles) of ethyl 4-methylpyrrol-3-carboxylate in 50 mL of benzene at room temperature under nitrogen atmosphere. The reaction mixture was stirred for 18 h. Water (100 mL) was added to the reaction mixture. The organic layer was separated, washed with brine and dried over anhydrous sodium sulfate. The solvent was removed and the residue was distilled giving 1.2 g (44%) of the title compound.
Preparation of 3.,4-Dimethylpyrrole-2-carboxaldehyde. To a solution of 0.92 mL (12 mmoles) of N,N-dimethylformamide in 10 mL of 1,2-dichloroethane was added dropwise 1.0 mL (12 mmoles) of phosphorus, oxychloride at 0°C. The ice-bath was removed and the reaction mixture was further stirred for 30 min. 3,4-Dimethylpyrrole (960.0 mg, 10 mmoles) was added to the above solution portionwise and the reaction mixture was stirred at 50°C for 5 h. The reaction mixture was poured into ice-cold 1:K sodium hydroxide solution (pH = 9 after mixing) and the resulting mixture was stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer w,3s extracted with ethyl acetate. The combined organic layer w,3s washed with brine until pH = 7, dried over anhydrous sodium sulfate and evaporated. The residue was chromatographed on a silica gel column eluting with a solvent mixture of ethyl acetate and hexane to afford 610 mg (50%) of the title compound.

A

3-[(3,4-i)imethylpyrrol-2-yl)methylene]-2-indolinone. A
reaction mixture of 67.0 mg (0.5 mmoles) of oxindole, 73.0 mg (0.6 mmoles) of the 3,4-dimethylpyrrole-2-carboxaldehyde, and 2 drops of piperidine in 2 mL of ethanol was stirred at 90°C
for 3 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 87.7 mg (37%) of the title compound, as a yellow solid.
5.11. Synthesis of 3-[(2,4-Dimethyl-3-l0 ethoxycarbonylpyrrol-5-yl)methylene]-2-indo7Linone A reaction mixture of 134.0 mg of oxindole, 234.3 mg of the 4-ethoxycarbonyl_-3,5-dimethylpyrrole-2-carboxaldehyde, and 3 drops of piperidine in 3 mL of ethanol was stirred at 90°C for 3 h. After cooling, the precipitate was filtered, l5.washed with cold ethanol, and dried to yield 244.6 mg (79%) of the title compound as a yellow solid.
5.12. Synthesis of 3-[(2,4 -Dimeth 1 yl)meahylene]-2-indolinone y pyrrol-5-20 A reaction mixture of 134.0 mg of oxindole, 147.8 mg of the 3,5-dimethyylpyrr~ole-2-carboxaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90°C for 3 h.
After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 136.7 mg (57%) of the title 25 compound as a :fellow solid.
5.13. Synthesis of 3-[(2-Methylmercaptothien-5-yl)methylene]-2-indolinone A reaction mixture of 134.0 mg of oxindole, 189.9 mg of 30 the 5-methylmercaptothiophene-2-carboxaldehyde, and 3 drops of piperidine :in 2 mL of ethanol was stirred at 90°C for 3 h.
After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 246.6 mg (900) of the title compound as a orange solid.
5.14. Synthesis of 3-[(2-Methylthien-5-yl)methylene]-2-indolinone A

~.'' ~ 2 %' 9 l .. , A reaction mixture of 134.0 mg of oxindole, 151.42 mg of the 5-methyltlZiophene-2-carboxaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90°C for 3 h.
After cooling, the precipitate was filtered, washed with cold 5 ethanol, and dried to yield 237.8 mg (99%) of the title compound as a yellow solid.
5.15. Synthesis of 3-[(3-Methylthien-2-yl)methylene]-2-indolinone 10 A reaction mixture of 134.0 mg of oxindole, 151.4 mg of the 3-methylthiophe:ne-2-carboxaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90°C for 3 h.
After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 157.8 mg (65%) of the title 15 compound as a yellow solid.
5.16. Syntlhesis of 3-(2,5-Dimethoxybenzylidenyl)-2-indo:linone 3-(2,5-L~imethoxybenzylidenyl)-2-indolinone is synthesized according to Method A.
5.17. Synthesis of 3-(2,3-dimethoxybenzylidenyl)-2-indo:Linone 3-(2,3-dimethoxybenzylidenyl)-2-indolinone is synthesized according to Method A.
5.18. Synthesis of 3-(3-bromo-6-methoxybenzylidenyl)-2-indolinone 3-(3-bromo-6-mEahoxybenzylidenyl)-2-indolinone is synthesized according to Method A.
5.19. Synthesis of 3-[4-(4-t-butylcarbonyl-piperazin-1-yl)benzylidenyl]-2-indolinone 3-[4-(4-t-butylcarbonyl-piperazin-1-yl)benzylidenyl]-2-indolinone is synthE~sized according to Method B.
5,20. Synthesis of 3-[(furan-2-yl)methylene]-2-indol.inone ~192i97 3-[(furan-2-yl)methylene]-2-indolinone is synthesized according to ZZethod A.
5.21. Synthesis of 3-(4-acetamidobenzylidenyl)-2-indolinone 3- (4-acei=amido:benzylidenyh) -2-indolinone is synthesized according to Method A.
5.22. Syntlhesis of 3-(2-chloro-4-hydroxybenzylidenyl)-2-indolinone 3_(2-chloro-4-lzydroxybenzylidenyl)-2-indolinone is synthesized according to Method A.
5.23. Syntlhesis of 3-(4-Bromobenzylidenyl)-2-indo:linone 3-(4-Bronobenzylidenyl)-2-indolinone is synthesized according to Method A.
5.24. Synthesis of 3-(4-Acetylaminobenzylidenyl)-2-indo:Linone 3-(4-Acet:ylaminobenzylidenyl)-2-indolinone is synthesized according to Method A.
5.25. Synthesis of 3-(2-Methoxybenzylidenyl)-2-indo:linone 3-(2-Methoxybenzylidenyl)-2-indolinone is synthesized according to Method A.
5.26. Synthesis of 3-(4-Dimethylaminobenzylidenyl)-1-mei~hyl-2-indolinone 3-(4-DimE~thylaminobenzylidenyl)-1-methyl-2-indolinone is synthesized according to Method A.
5.27. Synthesis of 3-(4-Dimethylaminobenzylidenyl)-2-indolinone 3-(4-Dime~thylaminobenzylidenyl)-2-indolinone is available from Maybridge Chemical Co. Ltd.

- ~ 1 ~2l ~~7 5.28. Synthesis of 3-(4-Bromobenzylidenyl)-1-methyl-2-iadolinone 3-(4-Bromobenzylidenyl)-1-methyl-2-indolinone is synthesized according to Method A.
5.29. Synthesis of 5-Chloro-3-(4-dimethylaminobenzylidenyl)-2-indolinone 5-Chloro--3-(4-dimethylaminobenzylidenyl)-2-indolinone is synthesized a<:cording to Method A.
5,30. Syntlhesis of 3-(4-Bromobenzylidenyl)-5-ahloro-2-indolinone 3-(4-Browobenz~~ilidenyl)-5-chloro-2-indolinone is synthesized according to Method A.
5.31. Syntlhesis of 3-(4-Diethylaminobenzylidenyl)-2-indo:linone 3- (4-Diet:hylam:inobenzylidenyl) -2-indolinone is synthesized according to Method A.
5.32. Synthesis of 3-(4-Di-n-buty:Laminobenzylidenyl)-2-indolinone 3-(4-Di-r~-buty:Laminobenzylidenyl)-2-indolinone is synthesized according to Method A.
5.33. Synthesis of 1-Methyl-3-[4-(morpholin-4-yl)be~nzylidenyl]-2-indolinone 1-Methyl-~3-[4-(morpholin-4-yl)benzylidenyl]-2-indolinone is synthesized according to Method B.
5.34. Synthesis of 5-Chloro-3-(4-(morpholine-4-yl)bE:nzylidenyl)-2-indolinone 5-Chloro-~3-(4-(morpholine-4-yl)benzylidenyl)-2-indolinone is synthesized according to Method B.
5.35. Synthesis of 3-(3,4-Dichlorobenzylidenyl)-2-indo7Linone 3-(3,4-Dichlorobenzylidenyl)-2-indolinone is synthesized according to Method A.

2?92797 5.36. Synthesis of 3-(2-Ethoxybenzylidenyl]-2-indo~linone 3-(2-Eth~~xybenzylidenyl]-2-indolinone is synthesized according to lKethod A.
5.37. Synthesis of 3-(4-Fluorobenzylidenyl)-2-indolinone 3-(4-Fluorobenzylidenyl)-2-indolinone is synthesized according to l~iethod A.
5.38. Synthesis of 3-[(Thien-2-yl)methylene]-2-indolinone 3-[(Thien-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.39. Synthesis of 3-(2-Methoxybenzylidenyl)-2-indolinone 3-(2-Metlaoxybenzylidenyl)-2-indolinone is synthesized according to Method A.
5~40. Synthesis of 3-[2-[3,5-Di-(tri;fluoromethyl)phenyl]furan-5-yl]methylene]-2 -i:ndolinone 3-[2-[3,5-Di-(trifluoromethyl)phenyl]furan-5-yl]methylene]--2 -indolinone is synthesized according to Method A.
5.41. Syntlhesis of 2,6-Di-(dimethylamino)-3,5-di-[(indolin-2-one-3-ylidenyl)met hyl]-phenylcyanide 2,6-Di-(dimethylamino)-3,5-di-[(indolin-2-one-3-ylidenyl)met hyl]-phenylcyanide is synthesized according to Method A.
5.42. Synthesis of 3-[(3-(2-carboxyethyl)-4-meth~~lpyrrol-5-yl)methylene]-2-indo linone 3-[(3-(2-~carbo:cyethyl)-4-methylpyrrol-5-yl)methylene]-2-indo linone is. synthesized according to Method A.

~ 3 ~~7 j7 5.43. Synthesis of 3-[(3,4-Dibromo-5-methylpyrrol-2-yl)m~ethylene]-2-indolinone 3-[(3,4-:Dibrom,o-5-methylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method B.
5.44. Synthesis of 3-[(3,4-Dimethyl-2-formylpyrrole-5-yl)methylene)-2-indolinone 3-[(3,4-l~imethyl-2-formylpyrrole-5-yl)methylene)-2-indolinone is synthesized according to Method A.
5,45. Synthesis of 3-{[4-(2-methoxycarbonylethyl)-3-methylpyrrol-5-yl]methylene }-2-indolinone 3-~[4-(2~-methoxycarbonylethyl)-3-methylpyrrol-5-yl]methylene :~-2-indolinone is synthesized according to Method A.
5.46. Synthesis of 3-[2-Iodofuran-5-yl)methylene]-2-indolinone 3-[2-Iodofuran-5-yl)methylene]-2-indolinone is synthesized according to Method A.
5.47. Synthesis of 3-[(3-Ethoxycarbonyl-2-methylfuran-5-yl)methylene]-2-indolin one 3-[(3-Ethoxyca:rbonyl-2-methylfuran-5-yl)methylene]-2-indolinone is synth~'sized according to Method A.
5,48. Synthesis of 3-[(3-Bromothiene-2-yl)m~ethylene]-2-indolinone 3-[(3-Bromothiene-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.49. Syntlhesis of 3-[(2-Chlorothiene-5-yl)m~sthylene)-2-indolinone 3-[(2-Chl.oroth:iene-5-yl)methylene)-2-indolinone is synthesized according to Method A.
5.50. Synthesis of 3-[(2,3-Dimethylfuran-5-yl)methylene]-2-indolinone 2i92i97 3-[(2,3-Dimethylfuran-5-yl)methylene]-2-indolinone is synthesized a~~cording to Method A.
5.51. Synthesis of 3-[(5-Nitrothien-2-yl)methylene]-2-in~dolinone 3-[(5-Nii~rothien-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.52. Synthesis of 3-[(2-Carboxythien-5-yl)methylene]-2-indolinone 3-[(2-Carboxythien-5-yl)methylene]-2-indolinone is synthesized according to Method A.
5.53. Synthesis of 3-[(2-Bromothiene-5-yl)methylene]-2-indolinone 3-[(2-Bromothiene-5-yl)methylene]-2-indolinone is synthesized according to Method A.
5.54. Synthesis of 3-[(4-Bromothiene-2-yl)meahylene]-2-indolinone 3-[(4-BromothiE:ne-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.55. Synthesis of 3-[(2-Sulphonylfuran-5-yl)meahylene]-2-indolinone sodium salt 3-[(2-Sulphony7_furan-5-yl)methylene]-2-indolinone sodium salt is synthesized according to Method A.
5.56. Synthesis of 3-[(Furan-2-yl)methylene]-2-indo7.inone 3-[(Furan-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.57. Synthesis of 3-[(2-Methylfuran-5-yl)meahylene]-2-indolinone 3-[(2-Methylfuran-5-yl)methylene]-2-indolinone is synthesized according to Method A.

5.58. synthesis of 3-[(2-Ethylfuran-5-yl)methylene-2-indolinone 3-[(2-Ethylfuran-5-yl)methylene-2-indolinone is synthesized according to Method A.
5.59. Synthesis of 3-[(2-Nitrofuran-5-yl)methylene]-2-indolinone 3-[(2-Nitrofuran-5-yl)methylene]-2-indolinone is synthesized according to Method A.
5,60. synthesis of 3-[(5-Hromofuran-2-yl)methylene]-2-indolinone 3-[(5-Bromofuran-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.6i. synthesis of 3-[(2-Ethylthien-5-yl)methylene]-2-indoliaone 3-[(2-Ethylthien-5-yl)methylene]-2-indolinone is synthesized according to Method A.
5.62. synthesis of 3-[(4,5-Dimethyl-3-ethylpyrrol-2-yl)methylene]-2-indolinone 3-[(4,5-Dimethyl-3-ethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.63. synthesis of 3-[(5-Ethouycarbonyl-4-ethouycarbonylethyl-3-ethoxycarbonylm ethylpyrrol-2-yl)methylene]-2-indolinone 3-[(5-Ethoxycarbonyl-4-ethoxycarbonylethyl-3-ethoxycarbonylmethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.64. synthesis of 3-[(5-Carboxy-3-ethyl-4-methylpyrrol-2-yl)methylene]-2-indoliaone 3-[(5-Carboxy-3-ethyl-4-methylpyrrol-2-yl)methylene]-2-~ndolinone is synthesized according to Method A.

- 2?92797 5.65. Synl:hesis of 3-[(3,5-Diiodo-4-methylpyrrol-2-yl)aaethylene]-2-indolinone 3-[(3,5-Diiodo-4-methylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.66. Synthesis of 3-[(5-Chloro-3-methoxycarbonyl-4-methoxycarbonylmethylpyrrol -2-yl)methylene]-2-indolinone 3-[(5-Chloro-3~-methoxycarbonyl-4-methoxycarbonylmethylpyrrol -2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.67. Synthesis of 3-[(3-Acetyl-5-ethoxycarbonyl-4-meth.ylpyrrol)-2-yl)methylene ]-2-indolinone 3-[(3-Acetyl-5-ethoxycarbonyl-4-methylpyrrol)-2-yl)methylene ]-2-indolinone is synthesized according to Method A.
5.68. Synthesis of 3-~[1-(3,5-Dichlorophenyl)pyrrol-2-yl]methylene}-2-indolinone 3-{[1-(3,5-Dichlorophenyl)pyrrol-2-yl]methylene}-2-indolinone is synthesized according to Method A.
5.69. Synthesis of 3-[1-(4-Chlorophenyl)pyrrol-2-yl)methylene]-2-indolinone 3-[1-(4-c:hloro;phenyl)pyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.70. Synthesis of 3-[(4-Ethoxycarbonyl-3-methyl)pyrrol-2-yl)methylene]-2-indolinone 3-[(4-Ethoxyca:rbonyl-3-methyl)pyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.71. Syntlhesis of 3-[(1-Methylpyrrol-2-yl)methylene]-2-indolinone 3-[(1-Met:hylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A.

' ~?9~~97 5.72. Synthesis of 3-[(5-Ethoxycarbonyl-3-etho:xycarbonylethyl-4-ethoxylcarbonyl methylpyrrol-2-yl)methylene]-2-indolinone 3-[(5-Ethoxyca:rbonyl-3-ethoxycarbonylethyl-4-ethoxylcarbonyl metlhylpyrrol-2-yl)methylene]-2-indolinone is synthesized ac:cordi:ng to Method A.
5.73. Synthesis of 3-[4-(Pyrrolidin-1-yl)benzylidenyl]-2-indolinone 3-[4-(Pyrrolid:in-1-yl)benzylidenyl]-2-indolinone is synthesized according to Method A.
5.74. Syntlhesis of 3-[(5-Methylimidazol-2 yl)methylene]-2-indolinone 3-[(5-Met:hylim:idazol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.75. Synthesis of 3-[(5-Methylthiazol-2 yl)methylene]-2-indolinone 3-[(5-Met:hylth_iazol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.76. Synthesis of 3-[(3-Methylpyrazol-5 yl)meahylene]-2-indolinone 3-[(3-Met:hylpyrazol-5-yl)methylene]-2-indolinone is synthesized according to Method A.
5.77. Synthesis of 3-[(Imidazol-4-yl)methylene]-2-indo7linone 3-[(Imida.zol-4--yl)methylene]-2-indolinone is synthesized according to Method A.
5.78. Synthesis of 3-[(4-Chloropyrazol-3 yl)methylene]-2-indolinone 3-[(4-Chloropyrazol-3-yl)methylene]-2-indolinone is synthesized according to Method A.

a 1 ~~%97 5.79. Synthesis of 3-[(4-Bromo-1-(4-chlo.robenzyl)pyrazol-5-yl)methylene]-2-indo - lino:ne 3-[(4-Bromo-1-(4-chlorobenzyl)pyrazol-5-yl)methylene]-2-indolinone is synthesized according to Method A.
5.80. Synthesis of 3-[(4-Chloro-1-methylpyrazol-3-yl)m~ethylene]-2-indolinone 3-[(4-Ch~Loro-1~-methylpyrazol-3-yl)methylene]-2-indolinone is synthesized according to Method A.
5.81. Syntlhesis of 3-[(4-Ethyl-3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone 3-[(4-Ethyl-3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method B.
5.82. Synthesis of 3-[(5-Ethylpyrrol-2-yl)methylene]-2-indolinone 3-[(5-Et:hylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method B.
5,83. Synthesis of 3-[3,5-Dimethyl-4-(propen-2-yl)pyrrol-2-yl)methylene]-2-indolinone 3-[3,5-Di.methy7L-4-(propen-2-yl)pyrrol-2-yl)methylene]-2-indolinone is synthEaized according to Method B.
5.84. Synthesis of 5,6-Dimethoxyl-3-[2,3-dimet:hoxylbenzylidenyl]-2-indolinone 5,6-Dimethoxyl--3-[2,3-dimethoxylbenzylidenyl]-2-indolinone is synthesized according to Method A.
5,g5. Synthesis of 3-[2,4,6-Trimethoxybenzylidenyl]-2-inclolinone 3-[2,4,6-Trimet:hoxybenzylidenyl]-2-indolinone is synthesized according to Method A.
5.86. Synthesis of 5-Chloro-3-[(pyrrol-2-yl)meahylene]-2-indolinone ?9?_~~7 5-Chloro-3-[(pyrrol-2-yl)methylene]-2-indolinone is synthesized a~~cording to Method A.
5.87. Synthesis of 5-Chloro-3-[(3-methylpyrrol-2-yl)methylene]-2-indolinone 5-Chloro~-3-[(3-methylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.88. Synthesis of 3-(4-isopropylbenzylidenyl)-2-indolinone 3-(4-isopropyl:benzylidenyl)-2-indolinone is available from MaybridgE~ Chemical Co. Ltd.
5.89. Synthesis of 5-Chloro-3-[(3,5-dimet:hylpyrrol-2-yl)methylene]-2-indo7.inone 5-Chloro-3-[(3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone is synthsaized according to Method A.
5.90. synthesis of 3-[(pyrrol-2-yl)methylene]-2-indol.inone -[(pyrrol-2-yl.)methylene]-2-indolinone is available from Maybridge Chemical Co. Ltd.
5.91. Synthesis of 5-Chloro-3-[(indol-3-yl)meahylene]-2-indolinone 5-Chloro-3-[(in.dol-3-yl)methylene]-2-indolinone is synthesized according to Method A.
5.92. Synthesis of 5-Chloro-3-[(thien-2-yl)methylene]-2-indolinone 5-Chloro-3-[(thien-2-yl)methylene]-2-indolinone is synthesized ac~~ording to Method A.
5.93. Synthesis of 5-Chloro-3-[(3-methylthien-2-yl)methylene]-2-indolinone 5-Chloro-:3-[(3-methylthien-2-yl)methylene]-2-indolinone is :synthesized according to Method A.

2_ 192 % 97 5.94. Synthesis of 5-Chloro-3-[(5-methylthien-2-yl)methylene]-2-indolinone 5-Chloro~-3-[(5-methylthien-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.95. Synthesis of 5-Chloro-3-[(5-ethylthien-2-yl)methylene]-2-indolinone 5-Chloro--3-[(5-ethylthien-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5, g6. Syntlhesis of 5-Chloro-3-[(5-methylmeraaptothien-2-yl)methylene]-2-indo:linone 5-Chloro-~3-[(5~-methylmercaptothien-2-yl)methylene]-2-indolinone i.s syni~hesized according to Method A.
5.97. Synthesis of 5-Chloro-3-[(imidazol-2 yl)meathylene]-2-indolinone 5-Chloro-3-[(imidazol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.98. Synthesis of 3-[2,4-Dimethoxy-6-methylbenzylidenyl]-2-indolinone 3-[2,4-Dimetho};y-6-methylbenzylidenyl]-2-indolinone is synthesized according to Method A.
5.99. Synthesis of 5-Nitro-3-[(pyrrol-2-yl)meahylene]-2-indolinone 5-Nitro-3-[(pyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.100. Synthesis of 3-[(3-Methylpyrrol-2-yl)me;thylene]-5-vitro-2-indolinone 3-[(3-Methylpyrrol-2-yl)methylene]-5-vitro-2-indolinone is ;synthesized according to Method A.
5.101. Synthesis of 3-[(3,5-Dimethylpyrrol-2-yl)methylene]-5-vitro-2-indolinone 2 ~ ~219T
3-[(3,5-I)imethylpyrrol-2-yl)methylene]-5-vitro-2-indolinone is synths=sized according to Method A.
5.102. Synthesis of 3-[(Indol-3-yl)methylene]-5-vitro-2-indolinone 3-[(Indol.-3-yl;Imethylene]-5-vitro-2-indolinone is synthesized according to Method A.
5.103. Synthesis of 5-Nitro-3-[(thien-2-yl)meahylene]-2-indolinone 5-Nitro-3-[(th:ien-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.104. Synthesis of 3-[(3-Methylthien-2-yl)meahylene]-5-vitro-2-indolinone 3-[(3-Methylthien-2-yl)methylene]-5-vitro-2-indolinone is synthsa ized according to Method A.
5.105. Synthesis of 3-[(5-Methylthien-2-yl)meahylene]-5-vitro-2-indolinone 3-[(5-Methylthi_en-2-yl)methylene]-5-vitro-2-indolinone is synthesized according to Method A.
5.106. Synthesis of 3-[(5-Ethylthien-2-yl)methylene]-5-vitro-2-indolinone 3-[(5-Ethylthie:n-2-yl)methylene]-5-vitro-2-indolinone is synthea ized according to Method A.
5.107. Synthesis of 3-[(5-Methylmercaptothien-2-yl)meahylene]-5-vitro-2-indolinone 3-[(5-Met:hylmercaptothien-2-yl)methylene]-5-vitro-2-indolinone is synthesized according to Method A.
5~108. Synthesis of 3-[(Imidazol-2-yl)methylene]-5-vitro-2-indolinone ' ! ~ ~~~~~~
3-[(Imida.zol-2--yl)methylene]-5-nitro-2-indolinone is synthesized according to Method A.
5.109. Synthesis of 3-[(Oxazol-2-yl)methylene]-2-indol.inone 3-[(Oxazol-2-yl.)methylene]-2-indolinone is synthesized according to Method A.
5.11o. Synthesis of 3-[(Oxazol-4-yl)methylene]-2-indol.inone l0 3-[(Oxazol-4-yl.)methylene]-2-indolinone is synthesized according to Method A.
5.111. Synth~,esis of 3-[(Oxazol-5-yl)methylene]-2-indolinone 3-[(Oxazol-5-yl)methylene]-2-indolinone is synthesized according to Method A.
5.112. Synthesis of 3-[(Thiazol-2-yl)methylene]-2-indolinone 3-[(Thiazol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5.113. Synthesis of 3-[(Thiazol-4-yl)methylene]-2-indolinone 3-[(Thiaz~~l-4-yl)methylene]-2-indolinone is synthesized ac~~ording to Method A.
5.114. Synthesis of 3-[(Thiazol-5-yl)methylene]-2-indolinone 3-[(Thiazol-5-yl)methylene]-2-indolinone is synthesized according to Method A.
5.115. Synthesis of 3-[(Imidazol-2-yl)methylene]-2-indolinone 3-[(Imida;Col-2-yl)methylene]-2-indolinone is synthesized according to Method A.

? ~~~~ ~i 5.116. Synthesis of 3-[(Pyrazol-3-yl)methylene]-2-indolinone 3-[(Pyra;.ol-3-yl)methylene]-2-indolinone is synthesized according to Method A.
5.117. synthesis of 3-[(Pyrazol-4-yl)methylene]-2-in~dolinone 3-[(Pyra~;ol-4-;yl)methylene]-2-indolinone is synthesized ac:cordi:ng to Method A.
l0 5,118. synthesis of 3-[(Isoxazol-3-yl)methylene]-2-indolinone 3-[(Isoxazol-3~-yl)methylene]-2-indolinone is synthesized according to Method A.
5.119. Syntlhesis of 3-[(Isoxazol-4-yl)methylene]-2-indolinone 3-[(Isoxazol-4~-yl)methylene]-2-indolinone is synthesized according to Method A.
5.120. Synthesis of 3-[(Isoxazol-5-yl)methylene]-2-indolinone 3-[(Isoxa.zol-5--yl)methylene]-2-indolinone is synthesized according to Method A.
5.121. Synthesis of 3-[(Isothiazol-3-yl)meahylene]-2-indolinone 3-[(Isoth.iazol--3-yl)methylene]-2-indolinone is synthesized according to Method A.
5.122. Synthesis of 3-[(Isothiazol-4-yl)meahylene]-2-indolinone 3-[(Isothiazol--4-yl)methylene]-2-indolinone is synthesized according to Method A.
5.123. Synthesis of 3-[(Isothiazol-5-yl)meahylene]-2-indolinone 3-[(Isothiazol-~5-yl)methylene]-2-indolinone is synthesized according to Method A.

~' ~ ',~L% ~?%
5.124. Synthesis of 3-[(1,2,3-Triazol-4-yl)methylene]-2-indolinone 3-[(1,2,3-Tria:Col-4-yl)methylene]-2-indolinone is synthesized according to Method A.
5.125. Synthesis of 3-[(1,3,4-Thiadiazol-2 yl)meahylene]-2-indolinone 3-[(1,3,4-Thiadiazol-2-yl)methylene]-2-indolinone is synthesized according to Method A.
5,126. S nthesis of 3 y -[(5-Phenyl-1,2,4-oxadiazol-3-yl)methylene]-2-indolinone 3-[(5-Phenyl-1,.2,4-oxadiazol-3-yl)methylene]-2-indolinone is synthesized according to Method A.
5.127. Synthesis of 3-[(3-Phenyl-1,2,4-oxadiazol-5-yl)methylene]-2-indolinone 3-[(3-Phenyl-1,2,4-oxadiazol-5-yl)methylene]-2-indolinone is synthEaized according to Method A.
5.128. Synthesis of 3-[(3-Phenyl-1,2,5-oxadiazol-4-yl)methylene]-2-indolinone 3-[(3-Phenyl-1,2,5-oxadiazol-4-yl)methylene]-2-indolinone is synthsaized according to Method A.
6 . EXAMPLES : In Vi: tro RTR Assays The following i:n vitro assays may be used to determine the level of activity and effect of the different compounds of the present invention on one or more of the RTKs. ~>imilar assays can be designed along the same lines for any tyrosine kinase using techniques 3 0 wel l known in the art .
6.1. Enzyme Linked Immunosorbent Assay (ELISA) Enzyme linked immunosorbent assays (ELISA) may be used to detect and measure the presence of tyrosine kinase activity. The ELISA may be conducted according to known protocols which are described in, for example, Voller, et al., 1980, "Enzyme-Linked Immunosorbent Assay," In': Manual of Clinical Immunology, 2d ed., edited by Rose and.Friedman, pp 359-371 Am. Soc. Of Microbiology, Washington, D.C.
The disclosed protocol may be adapted for determining activity with respect to a specific RTR. For example, the preferred protocols for conducting the ELISA
experiments for specific RTKs is provided below.
Adaptation of these protocols for determining a compound's activity for.other_members of the RTK family, ~
as well as non-receptor tyrosine kinases, are within the scope of those in the art.
6.1.1. FLR-1 ELISA
~ An ELISA assay was conducted to measure the kinase activity of the FLK-p receptor and more specifically, the inhibition or activation of protein tyrosine kinase activity on the FLK-1 receptor.:
Specifically, the following assay was conducted to measure kinase activity of the FLK-1 receptor in FLK-1/NIH3T3 cells.
Mater~Lals Aad Methods.
Materials. The following reagents and supplies were used:
a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96);
b. Cappel goat anti-rabbit IgG (catalog no.
. 55641);
c. PBS (Gibco Catalog No. 450-1300EB);
d, TBSW Buffer 50 mM Tris ( (pH 7.2), 150 mM NaCl and 0.1% TweenTM-20) ;
e. Ethanolamine stock (10% ethanolamine (pH,7.0), stored at 4°C);
f. HNTG buffer (20mM HEPES buffer (pH 7.5), 150mM
NaCl, 0.2o TritonTM X-100, and 10% glycerol);
g. EDTA (0.5 M (pH 7.0) as a 100X stock);

h. Sodium oritho vanadate (0.5 M as a 100X stock);
i. Sodi.um py:-o phosphate (0.2M as a 100X stock);
j. NUNC: 96 weall V bottom polypropylene plates (ApF~lied Scientific Catalog No. AS-72092);
k. NIH3T3 C7~~3 Cells (FLK-1 expressing cells);
1. DMEM: with 1X high glucose L Glutamine (catalog No. 11965--050) ;
m. FBS, Gibco (catalog no. 16000-028);
n, L-glutamine, Gibcp (catalog no. 25030-016);
o. VEGF, PeproTech, Inc. (catalog no. 100-20)(kept as 1 ~g/100 u1 stock in Milli-Q dH20 and stored at -20°C;
p. Affinity purified anti-FLK-1 antiserum, Enzymology Lab, Sugen, Inc.;
q. UB40 monoclonal antibody specific for phosphotymosine, Enzymology Lab, Sugen, Inc.
(see, Fendly, et al., 1990, Cancer Research 50:1550-1558);
r~ EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011);
s. 2,2-azino-~bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS~) solution (100mM citric acid (anhvydrous,) , 250 mM NazHP04 (pH 4. 0) , 0.5 mg/ml ABTS (Sigma catalog no. A-1888)), solution should be stored in dark at 4°C until ready for use;
t. H202 (30% ~;olution) (Fisher catalog no. H325) ;
u. ABTS,/HzOz (15m1 ABTS solution, 2 u1 Hz02) prep~~red 5 minutes before use and left at room temperature;
~. 0.2 1~I HC1 stock in HZO;
w. dimel:hylsulfoxide (100%)(Sigma Catalog No. D-8418); and y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).
Protocol. The following protocol was used for conducting the assay:

A

~~92~97 1. Coa~~ Corning 96-well elisa plates with 1.O~,g per well CappE:l Anti-rabbit IgG antibody in O.1M Na2C03 pH
9.6. Bring final volume to 150 ~,1 per well. Coat plates overnight at ~~°C. Plates can be kept up to two weeks when stored ai. 4 ° C .
2. Grow cells in Growth media(DMEM, supplemental with 2.OmM L-(ilutamine, 10% FBS) in suitable culture dishes until c:onflu~ent at 37°C, 5%
3. Harvest cells by trypsinization and seed in Corning 25850 polystyrene 96-well roundbottom cell plates, 25.00() cella/well in 200~c1 of growth media.
4. Grow cello at least one day at 37°C, 5% CO2.
5. Waste cell: with D-PBS 1X.
6. Add 200~C1,/well of starvation media (DMEM, 2.OmM
1-Glutamine, 0.1% FBS). Incubate overnight at 37°C, 5%
COZ .
7. Dilute Compounds/Extracts 1:20 in polypropylene 96 well plate=> using starvation media. Dilute dimethylsulfo~:ide 1:20 for use in control wells.
8. Remove starvation media from 96 well cell culture plate; and add 162 ~l of fresh starvation media to each well.
9. Add 18,1 of 1:20 diluted Compound/Extract dilution (from step 7) to each well plus the 1:20 dimethylsulfox:ide dilution to the control wells (+/-VEGF), for a final dilution of 1:200 after cell stimulation. Final dimethylsulfoxide is 0.5 %. Incubate the plate at 37°C, 5% COz for two hours.
10. Remove unbound antibody from ELISA plates by inverting plate to remove liquid. Wash 3 times with TBSW
+ 0.5% ethanolamine, pH 7Ø Pat the plate on a paper towel to remove excess liquid and bubbles.
11. Block plates with TBSW + 0.5% Ethanolamine, pH
7.0, 150 ~1 per well. Incubate plate thirty minutes while shaking on a microtiter plate shaker.
12. Wash plate 3 times as described in step 10.
13. Add 0.5~CC~/well affinity purified anti-FLU-1 polyclonal rabbit antiserum. Bring final volume to 150~,1/well with TBSW + 0.5o ethanolamine pH 7Ø
Incubate plate for thirty minutes while shaking.
14. Add 180 ~;1 starvation medium to the cells and stimulate cells with 20~1/well lO.OmM sodium ortho vanadate and 500 ng~/ml VEGF (resulting in a final concentration of l.OmM sodium ortho vanadate and 50ng/ml VEGF per well) for eight minutes at 37°C, 5% COz.
Negative control wells receive only starvation medium.
15. After eight minutes, media should be removed from the cells and washed one time with 200~,1/well PBS.
16. Lyse cells in 150~C1/well HNTG while shaking at room temperature for five minutes. HNTG formulation includes sodium ortho vanadate, sodium pyro phosphate and EDTA.
17. Wash ELIS.A plate three times as described in step 10.
18. Transfer cell lysates from the cell plate to elisa plate and incubate while shaking for two hours. To transfer cell lysat~e pipette up and down while scrapping the wells.
19. Wash plate three times as described in step 10.
20. Incubate '.ELISA plate with 0.02~Cg/well UB40 in TBSW + 05% ethanolamine. Bring final volume to 150~c1/well. I:ncubai~e while shaking for 30 minutes.
21. Wash plate three times as described in step 10.
22. Incubate ELISA plate with 1:10,000 diluted EIA
grade goat anti-mou:~e IgG conjugated horseradish peroxidase in TBSW -~ 0.5o ethanolamine, pH 7Ø Bring final volume to 150~i1/well. Incubate while shaking for thirty minutes.
23. Wash plate as described in step 10.
24. Add 100 ~,7_ of ABTS/H20z solution to well.
Incubate ten minute=> while shaking.
25. Add 100 ~Cl of 0.2 M HC1 for 0.1 M HC1 final to stop the color development reaction. Shake 1 minute at room temperature. Remove bubbles with slow stream of air and read the ELISA plate in an ELISA plate reader at 410 nm.
6.1.2. 8ER-2 ELISA
Assay 1: EGF Receptor-HER2 Chimeric Receptor Assay In Whole Calls. HER2 kinase activity in whole EGFR-NIH3T3 cells was measured as described below:
Mater~Eals cad Reageats. The following materials and reagents Were used to conduct the assay:
a. EGF: stock concentration= 16.5 ILM; EGF 201, TOYOBO, Co., Ltd. Japan.
b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain).
c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal)(see, Fendly, et al., supra).
d. Detection antibody: Goat anti-rabbit lgG horse radish peroxidase conjugate, TAGO, Inc., Burlingame, CA:
e. TBST buffer:
Tris-HCl, pH 7.2 50 mM
NaCl ~~ 150 mM
Trito~-300 0.1 f. HNTG 5X stock:
HEPES 0.1 M
NaCl 0.75 M
Glycerol 50%
Triton X-100 1.0%
g. ABTS stock:
Citric Acid 100 mM
Na2HP04 2 5 0 mM
3'0 HCl, conc. 0.5 pM
ABTS' 0.5mg/ml * _(2,2' -azinobis(3-ethylbenzthiazolinesulfonic acid)). Keep solution in dark at 4°C until use.
h~ Stock reagents of:
EDTA 100 mM pH 7.0 Na3V04 0.5 M

Na4 ( Pz07 ) 0 . 2 M
Procedure. The following protocol was used:
A. Pre-coat ELISA Plate 1. Coat ELISA plates (Corning 96 well, Cat.
#25805-96) with 05-101 antibody at 0.5 g per well in PBS, 100 ~1 final volume/well, and store overnight at 4°C.
Coated plates are good for up to 10 days when stored at 4°C.
2. On day of use, remove coating buffer and replace with 100 ~1 blocking buffer (5% Carnation- Instant Non-Fat Dry Milk in PBS). Incubate the plate, shaking, at room temperature (about 23°C to 25°C) for 30 minutes.
Just prior to use, remove blocking buffer and wash plate 4 times with TBST buffer.
B. Seedincr Cells 1. An NIH3T3 cell line overexpressing a chimeric receptor containing the EGFR extracellular domain and extracellular HER2 kinase domain can be used for this assay.
2. Choose dishes having 80-90% confluence for the experiment. Trypsinize cells and stop reaction by adding 10% fetal bovine serum. Suspend cells in DMEM
medium (10% CS DMEM medium) and centrifuge once at 1500 rpm, at room temperature for 5 minutes.
3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum) , and count the cells using trypan blue. Viability above 90% is acceptable. Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 ~1 per well, in a 96 well microtiter plate. Incubate seeded cells in 5% C02 at 37°C for about hours.
C. Assay Procedures 1. Check seeded cells for contamination using an inverted microscope. Dilute drug stock (10 mg/ml in 35 DMSO) 1:10 in DMEM medium, then transfer 5 1 to a TBST
well for a final drug dilution of 1:200 and a final DMSO

?v2%~i concentration of 1%. Control wells receive DMSO alone.
Incubate in 5 °> COZ at 3 7 ° C f or two hours .
2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of 10 ~C1 dilute EGF (1:12 dilution), 100 nM final concentration is attained.
3. Prepare fresh HNTG=sufficient for 100 ~1 per well; and place on ice.
HNTG' ( 10 ml ) HNTG stock 2.0 ml milli-Q H:20 7.3 ml EDTA, 100 mM, pH 7.0 0.5 ml Na3V04, 0.5 M 0.1 ml Na4 ( PzO~ ) , 0 . 2 M 0 . 1 ml 4. After 120 minutes incubation with drug, add prepared SGF ligand to cells, 10 ~,1 per well, to a final concentration of 100 nM. Control wells receive DMEM
alone. Incubate, shaking, at room temperature, for 5 minutes.
5. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG* to cells, 100 ~Cl per well.
Place on ice for 5 minutes. Meanwhile, remove blocking buffer from other ELISA plate and wash with TBST as described above.
6. With a pipette tip securely fitted to a micropipettor, scrape cells from plate and homogenize cell material by repeatedly aspirating and dispensing the HNTG'lysis buffer. Transfer lysate to a coated, blocked, and washed ELISA plate. Incubate shaking at room temperature for one hour.
7. Remove lysate and wash 4 times with TBST.
Transfer freshly diluted anti-Ptyr antibody to ELISA
plate at 100 u1 per well. Incubate shaking at room temperature fo:r 30 minutes in the presence of the anti-Ptyr antiserum (1:3000 dilution in TBST).
8. Remove the anti-Ptyr antibody and wash 4 times with TBS'r. 'transfer the freshly diluted TAGO anti-rabbit IgG antibody to the ELISA plate at 100 ~,1 per well. Incubate shaking at room temperature for 30 minutes (anti-rabbit IgG antibody: 1:3000 dilution in TBST).
9. Remove TAGO detection antibody and wash 4 times with TBST. Transfer freshly prepared ABTS/H20z solution to ELISA plate, 100 ~1 per well. Incubate shaking at room temperature for 20 minutes. (ABTS/HZOa solution: 1.0 ~1 30% H202in 10 ml ABTS stock)..
10. Stop reaction by adding 50 ~tl 5N H2S04 (optional), and determine O.D. at 410 nm.
11. The maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls.
Assail 2: HER-2-BT474 ELIBA. A second assay may be conducted to measure whole cell HER2 activity. Such assay may be conducted as follows:
Materials Aud Reagents. The following materials and reagents were used:
a. BT-474 (ATCC HBT20), a human breast tumor cell line which expresses high levels of HER2 kinase.
b~ Growth media comprising RPMI + 10% FBS + GMS-G
(Gibco supplement) + glutamine for use in growing BT-474 in an incubator with 5% C02at 37°C.
c. A monoclonal anti-HER2 antibody.
d. D-PBS:
KHZHP04 0.20 g/1 10 (GIBCO,310-4190AJ) K2HP04 2 .16 g/ 1 KC1 0.20 g/1 NaCl 8.00 g/1 (pH 7.2) e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-Fat Dry Milk).
f. TBST buffer:

Tris-HC1 50 mM
NaCl ~ 150 mM (pH 7.2, HC1 10 N) Triton x-loo 0.1%
wherein stock solution of TES (10X) is prepared, and TritonTM X-100 is added to the buffer during dilution.
g. HNTG buffer (5x) HEPES 0.1 M
NaCl 750 mM (pH 7.2 (HC1, 1 N) Glycerol 50%
TritonTM X-100 1~0%
Stock solution (5x) is prepared and kept in 4°C.
h. EDTA-HCl: 0.5 M pH 7.0 (10 N HC1) as 500X
stock:
i. Na~VO,: 0.5 M as 100X stock is kept at -80°C as aliquots.
j . Nay ( PZO, ) : 0 . 2 M as lOOX stock.
k. . Polyclonal antiserum anti-phosphotyrosine.
1. Goat anti-rabbit IgG, horseradish peroxidase (POD) conjugate (detection antibody), Tago (Cat. No. 4520; Lot No. i802): Tago, Inc., Burlingame, CA.
~m. ABTS solution:
Citric acid 100 mM
Na2HP0, 250 mM (pH 4.0, 1 N HC1) ABTS 0.5 mg/ml wherein ARTS is 2.2'-azinobis(3-ethylbenzthiazoline sulfonic acid). For this assay, the ABTS solution should be kept in the dark at 4°C. The solution should be discarded when it turns green.
n. Hydrogen peroxide: 30% solution is kept in dark - and 4°C.
Procedure. All the following steps are at room temperature and aseptically performed, unless stated °therwise. All ELISA plate washing is by rinsing with distilled water three times and once with TEST.

A. Cell Seeding 1: Grow BT474 cells in tissue culture dishes (Corning 25020-100) to 80-90% confluence and collect using Trypsin-EDTA (0.25%, GIBCO).
2. Resuspend the cells in fresh medium and transfer to 96-well tissue culture plates (Corninc~,~'~
25806-96) at about 25,000-50,000 cells/well (100 ~el/well) . Incubate the cells in 5% Co2at 37°C overnight.
B. ELISA Plate Coatinct and Blocking 1. Coat the ELISA plate (Corning 25805-96) with anti HER2 antibody at 0.5 ~.g/well in 150 ~l PBS
overnight at 4°C, and seal with parafilm. The antibody coated plates can be used up to 2 weeks, when stored at 4°C.
2. On the day of use, remove the coating solution, replace with 200 gel of Blocking Buffer, shake the plate, and then remove the blocking buffer and wash the plate just before adding lysate.
C. Assa~r Procedures 1. TBST the drugs in serum-free condition.
Before adding drugs, the old media is replaced with serum-free RPMI (90 ~tl/well) 2. Dilute drug stock (in 100% DMSO) 1:10 with RPMI, and transfer 10 ~Cl/well of this solution to the cells to achieve a final drug DMSO concentration at 1%.
Incubate the cells in 5% COZ at 37°C.
3. Prepare fresh cell lysis buffer (HNTG*) 5xHNTG 2 ml EDTA 0.2 ml Na3V04 0.1 ml Na4PZ0., 0. 1 ml H20 7 . 3 ml 4. After drug preincubation for two hours remove all the solution from the plate, transfer HNTG' (100 ul/well) to the cells, and shake for 10 minutes.
5. Use a 12-channel pipette to scrape the cells from the plate, and homogenize the lysate by repeat aspiration and dispensing. Transfer all the lysate to the ELISA plate and shake for 1 hour.
6. Remove the lysate, wash the plate, add anti-pTyr (1:3,000 with TBST) 100 ~1/well,-and shake for 30 minutes.
?. Remove anti-pTyr, wash the plate, add goat anti-rabbit IgG conjugated antibody (1:5,000 with TEST) 100 ~Cl/well, and shake for 30 minutes.
8. Remove anti-rabbit IgG antibody, wash the plate, and add fresh ABTS/FIzO= (1.2 ~Cl HzOZ to 10 ml ABTS) 10o ul/well to the plate to start color development, which usually takes 20 minutes.
9. Measure OD 410 nM, Dynatec~Mft5000.
6.1.3. PDGF-8 ELI6A
All cell culture media, glutamine, and fetal bovine serum were purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells were grown in a humid atmosphere. of ZO 90-95% air and 5-10% COz at 37°C. All cell lines were routinely subcultured twice a week and were negative for mycoplasma as determined by the Mycotect method (Gibco).
For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU) were grown to 80-90% confluency in growth medium (MErI with 10% FHS, NEAR, 1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in 0.5% serum at 25,000 to 30,000 cells per well. After overnight incubation in 0.5% serum-containing medium, cells were changed to serum-free medium and treated with test compound for 2 hr in a 5% COZ, 37°C incubator. Cells were then stimulated with ligand for 5-10 minutes followed' by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10%
glycerol, 5 mM EDTA, 5 mM Na3V04, 0.2% Triton'X-100, and 2 mM NaPyr). Cell Iysates (0.5 mg/well in PBS) were transferred to ELISA plates previously coated with receptor-specific antibody and which had been blocked with 5% milk in TBST (50 mM Tris-HC1 pH 7.2, 150 mM NaCl and 0.1% Triton~X-100) at room temperature for 30 min.
Lysates were incubated with shaking for 1 hour at room temperature. The plates were washed with TBST four times and then incubated with polyclonal anti-phosphotyrosine antibody at room temperature for 30 minutes. Excess anti-phosphotyrosine antibody was removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG
antibody was added to the ELISA plate for 30 min at room temperature followed by rinsing with TBST four more times. ABTS (100 mM citric acid, 250 mM Na2HP04 and 0.5 mg/mL 2,2~-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H2O2 (1.2 mL 30% Hz02 to 10 ml ABTS) was added to the ELISA plates to start color development.
Absorbance at 410 nm with a reference wavelength of 630 nm was recorded about 15 to 30 min after ABTS addition.
6.1.9. IGF-I EhISA
The following protocol may be used to measure phosphotyrosine level on IGF-I receptor, which indicates IGF-I receptor tyrosine kinase activity.
Materials And Reagents. The following materials and reagents were used:
a. The cell line used in this assay is 3T3/IGF-1R, a cell line which overexpresses IGF-i receptor.
b. NIH3T3/IGF-1R is grown in an incubator with 5%
C02 at 37°C. The growth media is DMEM + 10% FBS
(heat inactivated)+ 2mM L-glutamine.
c. Anti-IGF-iR antibody named 17-69 is used.
Antibodies are purified by the Enzymology Lab, SUGEN, Inc.
d. D-PBS:
KHzP04 0.20 g/1 KZHP04 2.16 g/1 KC1 0.20 g/1 NaCl 8.00 g/1 (pH 7.2) e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-Fat Dry Milk) f. TBST buffer:

Tris-HC1 50 mM
NaCl.~ 150mM (pH 7.2/HCl 10N) Triton X-100 0.1%
Stock solution of TBS 110X) is prepared, and TritonTM X-100 is added to the buffer during dilution.
g . HNTG buf f er HEPES 20 mM
NaCl 150 mM (pH 7.2/HC1 1N) Glycerol 10%
TritonTM X-100 0.2%
Stock solution (5X) is prepared and kept at 4°C.
h. EDTA/HC1: 0.5 M pH 7.0 (NaOH) as 100X stock.
i. Na3V04: 0.5 M as lOOX stock and aliquots are kept in -80°C.
j . Na4Pz07: 0. 2 M as 100X stock.
k. Insulin-like growth factor-1 from Promega (Cat, G5111).
1~ Polyclonal antiserum anti-phosphotyrosine:
rabbit sera generated by Enzymology Lab., SUGEN
Inc.
m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (Cat. No. 4520, Lot No. 1802):
Tago, Inc., Burlingame, CA.
n. ABTS (2.2~-azinobis(3-ethylbenzthiazolinesulfonic acid)) solution:
Citric acid 100 mM
Na2HP04 w250 mM (pH 4.0/1 N HC1) ABTS 0.5 mg/ml STS solution should be kept in dark and 4°C. The solution should be discarded when it turns green.
o. Hydrogen Peroxide: 30% solution is kept in the dark and at 4°C.
Procedure. All the following steps are conducted at room temperature unless it is specifically indicated.
All ELISA plate washings are performed by rinsing the plate with tap water three times, followed by one TBST
rinse. Pat plate dry with paper towels.
A. Cell Seeding:
1. The cells, grown in tissue culture dish (Corning 25020-100) to 80-90% confluence, are harvested with Trypsin-EDTA (0.25%, 0.5 ml/D-100, GIBCO).
2. Resuspend~the cells in fresh DMEM + 10%
FBS + 2mM L-Glutamine, and transfer to 96 - well tissue culture plate (Cornir~ 25806-96) at 20,000 cells/well .
(100 ~Cl/well). Incubate for 1 day then replace-medium to serum-free medium (90/1) and incubate in 5% COz and 37°CI
overnight. .
B. ELISA Plate Coating and Blocking:
1. Coat the ELISA.plate (Corning 25805-96) i5 with Anti-IGF-1R Antibody at 0.5 ~g/well in 100 ~tl PBS at .
least 2 hours.
2. Remove the coating solution, and replace with 100 ~l Blocking Buffer, and shake for 30 minutes.
Remove the blocking buffer and wash the plate just before adding.lysate.
C. Assay Procedures:
1. The drugs are tested in serum-free condition.
2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-well poly-propylene plate, and transfer 10 ~1/well of this solution to the cells to achieve final drug dilution 1:100, and final DMSO concentration of 1.0%. Incubate the cells in 5%wCO, at 37°C for 2 hours.
3... Prepare fresh cell lysis buffer (HNTG*) ~, HNTG 2 ml EDTA 0.1 ml Na3V0,, 0.'1 ml Nas (PzO~) 0.1 ml Hz0 7.3 ml 4. After drug incubation for two hours, transfer 10 ~t1/well of 200nM IGF-1 Ligand .in PBS to the cells (Final Conc. - 20 nM), and incubate at 5% C02 at 37°C for 10 minutes.
5. Remove media and add 100~1/well HNTG* and shake for 10 minutes. Look at cells under microscope to see if they are adequately lysed.
6. Use a 12-channel pipette to scrape the cells from the plate, and homogenize the lysate by repeat aspiration and dispense. Transfer all the lysate to the antibody coated ELISA plate, and shake for 1 hour.
7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000 with TBST) 100 ~Cl/well, and shake for 30 minutes.
8. Remove anti-pTyr, wash the plate, transfer Tago (1:3,000 with TBST) 100 ~ul/well, and shake for 30 minutes:
9. Remove detection antibody, wash the plate, and transfer fresh ABTS/H2O2 (1.2 ~1 Hz02 to 10 ml ABTS) 100 ~cl/well to the plate to start color development.
10. Measure OD in Dynate5000, which is connected to Ingres~
6.1.5. EaF Receptor ELIBA
EGF Receptor kinase activity (EGFR-NIH3T3 assay) in whole cells was measured as described below:
Materials aad Reagents. The following materials and reagents were used a. EGF Ligand: stock concentration = 16.5 ;CM; EGF
201, TOYOBO, Co., Ltd. Japan.
3o b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain).
c. Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).
d. Detection antibody: Goat anti-rabbit 1gG horse .radish peroxidase conjugate, TAGO, Inc., Burlingame, CA.
e. TBST buffer:

Tris-HC1, pH 7 50 mM
NaCl T~ 150 mM' Triton ~-100 0.1 f. HNTG 5X stock:
HEPES 0.1 M
NaCl 0.75 M
Glycerol 50 TritonTM X-100 1.0%
g. ABTS stock:
Citric Acid 100 mM
Na2HP0~ 250 mM
HC1, conc. 4.0 pH
ABTS' 0.5 mg/ml Keep solution in dark at 4°C until used.
h. Stock reagents of:
EDTA l00 mM pH 7.0 Na3V0,, 0.5 M
Na4 (PaO~) 0. 2 M
Procedure. The following protocol was used:
A. Pre-coat ELISA Piate 1. Coat ELISA plates (Cornin96 well, Cat.
X25805-96) with 05-101 antibody at 0.5 ~g per well in FBS, 150 gel final volume/well, and store overnight at 4°C. Coated plates are good for up to 10 days when stored at 4°C.
2. On day of use, remove coating buffer and replace with blbcking buffer (5% Carnatio~~Instant NonFat Dry Milk in PBS). Incubate the plate, shaking, at room temperature (about 23°C to 25°C) for 30 minutes. Just prior to use, remove blocking buffer and wash plate 4 3o times with TBST buffer.
B. Seedinq Cells 1. NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987) can be use for this assay.
2. Choose dishes having 80-90% confluence for the experiment. Trypsinize cells and stop reaction by adding 10% CS DMEM medium. Suspend cells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1000 rpm, and once at room temperature for 5 minutes.
3. Resuspend cells in seeding medium'(DMEM, 0.5% bovine serum), and count the cells using trypanT~
blue. Viability above 90% is acceptable. Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100' l per well, in a 96 well microtiter plate. Incubate seeded cells in 5% COz at 37°C for about 40 hours.
C. Assav Procedures.
1. Check seeded cells for contamination using an inverted microscope. Dilute drug stock (l0~mg/ml in DMSO) 1:10 in DMEM medium, then transfer~5 ~1 to a test well for a final drug dilution of 1:200 and a final DMSO
concentration of 1%. Control wells receive DMSO alone.
Incubate in 5% COz at 37°C for one hour.
2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of 10 ~1 dilute EGF (1:12 dilution), 25 nM final concentration is attained..
3. Prepare fresh 10 ml HNTG' sufficient for 100 ~C1 per well wherein HNTG* comprises: HNTG stock (2.0 ml), milli-Q Hz0 (7.3 ml), EDTA, 100 mM, pH ?.0 (0.5 ml), Na3V04 0.5 M (0.1 ml) and, Na,, (PzO.,) , 0.2 M (0.1 ml) .
4. Place on ice.
5. After two hours incubation with drug, add prepared EGF ligand to cells, 10 ~l per well, to yield a final concentration of 25 nM. Control wells receive DMEM
alone. Incubate, shaking, at room temperature, for 5 minutes.
6. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG'to cells, 100 ~C1 per well.
Place on ice for 5 minutes. Meanwhile, remove blocking buffer from other ELISA plate and wash with TBST as described above.
7. With a pipette tip securely fitted to a micropipettor, scrape cells from plate and homogenize cell material by repeatedly aspirating and dispensing the L ~ 92~~91 HNTG*lysis buffer. Transfer lysate to a coated, blocked, and washed EhISA plate. Incubate shaking at room temperature f:or one hour.
8. Remove lysate and wash 4 times with TBST.
Transfer fre~~hly diluted anti-Ptyr antibody to ELISA
plate at 100 ~,1 per well. Incubate shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiseru~,m (1:3000 dilution in TBST).
9. Remove the anti-Ptyr antibody and wash 4 times with TH~ST. Transfer the freshly diluted TAGO 30 anti-rabbit IgG antibody to the ELISA plate at 100 ~C1 per well. Incubate shaking at room temperature for 30 minutes (anti-rabbit IgG antibody: 1:3000 dilution in TBST).
10. Remove detection antibody and wash 4 times with TBST. Transfer freshly prepared ABTS/Hz02 solution to ELISA plate, 100 ~cl. per well. Incubate at room temperature for 20 minutes. ABTS/Hz02 solution: 1.2 ~C1 3 0% H20z in 10 ml ABTS stock .
11. Stop reaction by adding 50 ~,1 5N HZS04 (optional), and determine O.D. at 410 nm.
12. The maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls.
6.1.6. Cellular Insulin Receptor ELISA
The following protocol was used to determine whei~her the compounds of the present invention possessed insulin receptor tyrosine kinase activity.
Material: And Reagents. The following materials and reagents were used to measure phophotyrosine levels on the insulin rEaceptor (indicating insulin receptor tyrosine kinase activity):

1. The preferred cell line was an NIH3T3 cell line (ATCC No. 1658) which overexpresses Insulin Receptor (H25 cells);
2. H25 cells are grown in an incubator with 5% C02 at 37°C. The growth media is DMEM + 10% FBS (heat inactivated) + 2mm L-Glutamine;
3. For ELISA plate coating, the monoclonal anti-IR
antibody named BBE is used. Said antibodies was purified by the Enzymology Lab, SUGEN, Inc.;
4. D-PBS, comprising:
KH2PO4 0.20 g/1 (GIBCO, 310-4190AJ) K2HP04 2 .16 g/ 1 KC1 0.20 g/1 NaCl 8.0O g/1 (pH 7.2);
5. Blocking Buffer: TBST plus 5% Milk (Carnatio~i Instant Non-Fat Dry Milk);
6. TBST buffer, comprising:
Tris-HC1 50mM
NaCl 150mM pH 7.2 (HC1, 1 N) Trito~X-100 0.1%
Note: Stock solution of TBS (10X) is prepared, and Triton X-100 is added to the buffer during dilution;
7. HNTG buffer, comprising:
HEPES 20mM
NaCl 150mM pH 7.2 (HC1, 1 N) Glycerol 10%
Tritdn ~X-100 0.2%
Note: Stock solution (5X) is prepared and kept at 4°C;
8. EDTA.HC1: 0.5 M pH 7.0 (NaOH) as 100X stock;
9. Na3V04: 0.5 M as 100X stock and aliquots are kept in -80°C;
10. Na4P20.,: 0.2 M as 100X stock;
11. Insulin from GIBCO BRL (Cat# 18125039);
12. Polyclonal antiserum Anti-phosphotyrosine:
rabbit sera generated by Enzymology Lab., SUGEN Inc.;

13. Detection antibody, preferably goat anti-rabbit IgG, POD.conjugate, Tago (Cat. No..4520: Lot No. 1802):
Tago, Inc., Hurlingame, CA;
14. ABTS solution, comprising:
Citric acid 10o mM
NaZHPO~ 250 mM pH 4. 0 ( 1 N I;CI) ABTS 0.5 mg/ml wherein ABTS is 2,2'-azinobis (3-ethylbenathiazoline sulfonic acid) and stored in the dark at 4°C and discarded when it turns green;
15. Hydrogen Peroxide: 30% solution is kept iw thel ..
dark and at 40°C.
Protoco3. All the following steps are conducted at room temperature unless it is specifically indicated.
i5 All ELISA plate washings are perfonaed by rinsing the plate with tap water three times, followed by one TEST
rinse. All plates were tapped dry with paper towels -prior to use.
A. Cell Seeding:
Z0 1. The cells were grown in tissue culture dish (10 cm, Cornin~ 25020-100) to 80-90% confluence and harvested with Trypsin-EDTA (0.25%, 0.5 ml/D-100, GIBCO);
2. Resuspend the cells in fresh DMEM + 10%
FBS + 2mM L-Glutamine, and transfer to 96 - well tissue 25 culture plate (Corni~~~;s 25806-96) at 20,000 cells/well (100 ~l/well). The cells are then incubated for d day.
Following such incubation, 0.01% serum medium (90/1) replaces the old media and the cells incubate in 5% COZ
and 37°C overnight.
30 B. ELISA Plate Coating and Blocking:
1. Coat the ELISA plate (Cornin~i25805-96) with Anti-IR Antibody at 0.5 ~g/well in 100 ~1 PBS at least 2 hours:
2. Remove the coating solution, and replace 35 with 100 ~cl; blocking Buffer, and shake for 30 minutes.
Remove the blocking buffer and wash the plate just before adding lysate.

._ . ~ ~92T~7 C. Assay Procedures 1. The drugs are tested in serum-free condition.
2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-well poly-propylene plate, and transfer 10 ~1/well of this solution to the cells to achieve final drug dilution 1:100, and final DMSO concentration of 1.0%. Incubate the cells in 5% COz at 37°C for 2 hours.
3. Prepare fresh cells lysis buffer (HNTG*) HNTG (5x) 2 ml EDTA 0.1 ml NajV04 0.1 ml Na,~P,O., 0.1 ml HZU 7.3 ml HNTG* 10 ml 4. After drug incubation for two hours, transfer 10 ~cl/well of 1~CM insulin in PBS to the cells (Final concentration = 100 nM), and incubate at 5% COZ at 37°C for 10 minutes.
5. Remove media and add 100 ~,1/well HNTG* and shake for 10 :minutes. Look at cells under microscope to see if they are adequately lysed.
6. Using a 12-channel pipette, scrape the cells from the plate, and homogenize the lysate by repeat aspiration and dispense. Transfer all the lysate to the antibody coated ELISA plate, and shake for 1 hour.
7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000 with TBST) 100 ~,1/well, and shake for 30 :minutes.
8. Remove anti-pTyr, wash the plate, transfer Togo (1:3,000 with TBST) 100 ~.1/well, and shake for 30 minutes.
9. Remove detection antibody, wash the plate, and transfer fresh ABTS/H202 (1.2 ~,1 Hz02 to 10 ml ABTS) 100 ~1/well to the plate to start color development.

10. Measure OD in Dynatec~MR5000, which is connected to Ingres7M All following steps should follow Ingre~instruction.
6.1.7. Experimental Results From ELISA
Assays The experimental results for various compounds according to the invention using the above-described protocols are set forth at Table 1:

ELISA Assay Results Kinase (Example) IC50 (~rM)IC50 (/~M)IC50 (~rM)IC50 (~M) C50 (~tM) 27 19.4 0.8 4313 14.5 18.8 11 16.9 8.0 1B 12 0.39 16 87.4 4.2 17 11.8 20 28.8 22 2.2 24 8.5 26 22.6 28 22.5 29 7.9 11.2 32 20.9 4 33.1 2.1 33 21.6 39.4 34 4.1 5 5.8 1.6 90.2 36 4 ' 51.5 37 9.6 38 4.7 39 14.8 36.7 41 6.4 8 2.9 89.8 42 0.4 43 1.8 9 17 0.24 44 23.8 10 0.17 45 53.7 1.1 11 0.07 12 10.8 0.11 -. -3 4g 15 . 4 13 2.3 50 4.6 14 2.4 53 51.4 15 4.5 70.6 55 8.6 57 73.4 58 41.2 _ 77 _ - 2?92197 (Example) IC50 (~M) IC50 (feM)IC50 Rinase (/!M) IC50 (~.iM)IC50 (/.tM) 59 22.8 6 4.5 92.6 61 3.4 44 62 65.5 0.14 64 36.2 70 - 0.18 71 20.3 73 86 1.6 74 55.9 2.7 76 8.7 77 14.2 1.5 78 7.4 81 0.15 7 5.3 39.6 30.4 6.2. Cel:L Growth Assays The following assays may be conducted to measure the ej=fect of the claimed compounds upon cell growth as a rE~sult of the compound's interaction with one or more RTKs. Unless otherwise specified, the following assays may be generally applied to measure the activity of a compound against any particular RTK. To the extent that an assay, set forth below, refers to a specific RTK, one skilled in the art would be able to adapt the disclosed protocol for use to measure the activity of a second RTK.
6.2.1. Soft Agar Assay The soft agar assay may be used to measure the effects of substances on cell growth. Unless otherwise stated the soft agar assays were carried out as follows:
Material And Reagents. The following materials and reagents were used:
a, A water bath set at 39°C and another water bath at 37°C.
_ 78 _ b. 2X assay medium is comprised of 2X Dulbecco's ~
SModified Eagle's Medium (DMEM) (Gibco Cat.
CA400-4AN03) supplemented by the following:
~ 20% Fetal Bovine Serum (FBS) 2 mM
sodium pyruvate 4 mM glutamine amine;
and ~ 20 mM HEPES Non-essential Amino Acids (1:50 from lOOx stock).
c. 1X assay medium made of 1X DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM
glutamine, 10 mM HEPES, non-essential amino acid (1:100 from lOOx stock).
d. 1.6% SeaPlaque Agarose in autoclave bottle.
e: Sterile 35 nun Corning plates (FMC Bioproducts Cat. #50102) f. Sterile 5 ml glass pipets (individually wrapped).
g. Sterile l5 ml and 50 ml conical centrifuge tubes.
h. Pipets and sterile tips.
2o i, Sterile microcentrifuge tubes.
j. Cells in T75 flasks: SKOV-3 (ATCC HTB77).
k. 0.25% Trypsin solution (Gibco #25200-015).
Procedure. The following procedure was used to conduct the soft agar assay:
A. Procedure for making the base layer 1. Have all the media warmed up in the 37°C
water bath.
2. To make 1X of assay medium + 0.8% agar:
make a 1:2 (vol:vol) dilution of melted agar (cooled to 39°C), with 2X assay medium.
3. Keep all media with agar warm in the 39'C
water bath when not in use.
4. Dispense 1 ml of 1X assay medium + 0.8%
agar into dishes and gently swirl plate to form a uniform base layer. Bubbles should be avoided.

5. Refrigerate base layers to solidify (about 20 minutes). Base layers can be stored overnight in the refrigerator.
B. Pro~~edure for collectingr cells 1. Take out one flask per cell line from the incubator; as~~irate off medium; wash once with PBS and aspirate off; add 3 ml of trypsin solution.
2. After all cells dissociate from the flask, add 3 ml of 1:~ assay media to inhibit trypsin activity.
Pipet the cells up and down, then transfer the suspension into a 15m1 tube.
3. Determine the concentration of cells using a Coulter counter, and the viability by trypan blue exclusion.
4. Take out the appropriate volume needed to seed 3300 viable cells per plate and dilute it to 1.5 ml with 1X assay medium.
C. Procedure for makinct the upper 0 4% ag~arose layer:
1. Add TBST compounds at twice the desired final assay concentration; + 1.5 ml of cell suspension in 1X assay medium 10% FBS; + 1.5 ml of 1X assay medium +
0.8% agarose': Total. = 3.0 ml 1X media 10% FBS + 0.4%
agarose with 3300 viable cells/ml, with and without TBST
compounds.
*(Made by 1:2 dilution of 2X media with 1.6% agar 30 for the base layer procedure above.) 2. Plate 1 ml of the Assay Mix onto the 1 ml base layer. The duplicates are plated from the 3 ml volume.
3. Incubate the dishes for 2-3 weeks in a 100% humidified, 10% COZ incubator.
4. Colonies that are 60 microns and larger are scored positive.

2192797 ~.
6.2.2. Sulforhodamine B (SRB) Growth Assays The SRB assays may be used to measure the effects of substances on cell growth. The assays are carried out as follows:
Assay 1: 3T3/E/H+TGF-a(T) Cell Growth SRH Assay Materials:
96-well flat bottom sterile plates 96-well round x~ottom sterile plates sterile 25 ml or 100 ml reservoir pipets, multi-channel pipetman sterile pipet taps sterile 15 ml a.nd 50 ml tubes Reagents:
0.4% SRB in 1% acetic acid 10 mM Tris bass:
10% TCA
1% acetic acid sterile DMSO (:>igma) compound in DMaO (100 mM or less stock solution) 25% Trypsin-EDTA in Cell Dissociation Solution (Sigma) Cell line and growth medium:
3T3/E/H+TGF-a(f) (NIH 3T3 clone 7 cells expressing EGF-R/HER2 chimera and TGF-a, tumor-derived autocrine loop cells) 2% calf serum/I)MEM + 2 mM giutamine Protocol:
Day 0: Cell P:Lating:
This part of a:~say is carried out in a laminar flow hood.
1. Tryps:inize cells as usual. Transfer 100 ~cl of cell suspensio~z to 10 ml of isotone. Count cells with the Coulter Counter.

2. Dilute cells in growth medium to 60,000 cells/ml. Transfer 100 ~1 of cells to each well in a 96-well flat bottom plate to give 6000 cells/well.
3. Use half of plate (4 rows) for each compound and quadruplicate wells for each compound concentration, a set of 4 wells for medium control and 4 wells for DMSO
control.
4. Gently shake plates to allow for uniform attachment of the cells.
5. Incubate the plates at 37°C in a 10% C02 incubator.
Day 1: Addition of Compound:
This part of assay is carried out in a laminar flow hood.
1. In 96 well-round bottom plate, add 125 ~l of growth medium to columns 3 to 11. This plate is used to titrate out the compound, 4 rows per compound.
2. In a sterile 15 ml tube, make a 2X solution of the highest concentration of compound by adding 8 ~1 of the compound vto a total of 2 ml growth medium for a dilution of 1:250. At this dilution, the concentration of DMSO is 0.~~% for a 2X solution or 0.2% for 1X solution on the cells. The starting concentration of the compound is u:~ually 100 uM but this concentration may vary depending upon the solubility of the compound.
3. Tran:~fer the 2X starting compound solution to quadruplicate wells in column 12 of the 96-well round bottom plate. Do 1:2 serial dilutions across the plate from right to left by transferring 125 ~1 from column 12 to column 11, column 11 to 10 and so on. Transfer 100 ~1 of compound dilutions onto 100 ~1 medium on cells in corresponding wells of 96-well flat bottom plate. Total volume per well should be 200 ~1.
4. For ~rehicle control, prepare a 2X solution of DMSO at 0.4% I)MSO in growth medium. Transfer 100 ~1 of the DMSO solution to the appropriate wells of cells. The final concentration of DMSO is 0.2%.
5. For the medium control wells, add 100 ~1/well of growth medium to the appropriate wells of cells.
6. Return the plate to the incubator and incubate for 4 days.
Day 5: Development of Assay This part of assay is carried out on the bench.
1. Aspirate or pour off medium. Add 200 ~1 cold.
10% TCA to each well to fix cells. Incubate plate for at least 60 min. at 4°C.
2. Discard TCA and rinse wells 5 times with water.
Dry plates upside down on paper towels.
3. Stain cells with 100 ~,1/well 0.4% SRB for 10 min.
4. Pour off SRB and rinse wells 5 times with 1%
acetic acid. Dry plates completely upside down on paper towels.
5. Solubilize dye with 100 ~C1/well 10 mM Tris base for 5-10 min. on shaker.
6. Read plates on DynatechMELISA Plate Reader at 570 nm with reference at 630 nm.
Assay 2: 3T3/EGF-R+TGF-a(T) Cell Growth SRB Assay Materials and Reagents same as for Assay 1.
Cell line and growth medium:
3T3/EGF-R+TGF-a(T) (NIH 3T3 clone 7 cells expressing EGF-R and TGF-a, tumor-derived autocrine loop cells) 2% calf serum/DMEM + 2 mM glutamine Protocol:
Day 0: Cell Plating:
This part of assay is carried out in a laminar flow hood.

1. Trypsinize cells as usual. Transfer 100 ~C1 of cell suspension to 10 ml of isotone. Count cells with the Coulte~~ounter.
2. Dilute cells in growth medium to 60,000 cells/ml. Transfer 100 ~1 of cells to each well in a 96-well flat bottom plate to give 6000 cells/well.
3. Use half of plate (4 rows) for each compound and quadruplicate wells for each compound concentration, a set of 4 wells for medium control and 4 wells for DMSO
control.
4. Gently shake plates to allow for uniform attachment of the cells.
5. Incubate the plates at 37°C in a 10% C02 incubator.
Day 1: Addition of Compound: same as for Assay 1.
Day 5: Development of Assay: same as for Assay 1.
Assay 3: 3T3/PDGF-~BR/PDGF-BB(T) Cell Growth BRH Assay Cell line and arowth medium:
3T3/PDGF-/iR/PDGF-BB(T) (NIH 3T3 clone 7 cells expressing PDGF (3-receptor and PDGF-BB, from tumors resected from athymic mice) 2% calf serum/DMEM + 2 mM glutamine Protocol:
Day 0: Cell PlatincL
This part of assay is carried out in a laminar flow hood.
1. Trypsinize cells as usual. Transfer 200 ~tl of cell suspension to 10 ml of isotone. Count cells on the Coulter Counter.
2. Dilute cells in growth medium to 60,000 cells/ml. Transfer 100 ~1 of cells to each well in a 96-well flat bottom plate to give 6000 cells/well.

3. Allow half of plate (4 rows) for each compound and quadruplicate wells for each compound concentration, a set of 4 wells for medium control and 4 wells for DMSO
control.
4. Gently shake plates to allow for uniform attachment of the cells to the plate.
5. Incubate the plates at 37°C in a 10% COz incubator.
Day 1: Addition of Compound: same as for Assay 1.
Day 5: Development of Assay: same as for Assay 1.
Assay 4: Human Smooth Muscle Cells (SMC) Groarth 8RB
Assay Materials and Reagents same as for Assay 1:
Cell line and growth medium:
Human Aortic Smooth Muscle cells (Clonetics Clonetics's~Bullet Kit: Smooth Muscle Basal Medium (SmBM) which is modified MCDB 131 containing fetal bovine serum (5%), hFGF (2ng/ml), hEGF (0.1 ng/ml), insulin (5.0 ug/ml), gentamicin (50ug/ml) and amphotericin B (50 ng/ml) Protocol:
Day 0: Cell plating:
This part of assay is carried out in a laminar flow hood.
1~ Trypsinize cells as usual. Transfer 200 ~,1 of cell suspension to 10 ml of isotope. Count cells on the Coult r~~Counter.
2. Dilute cells in growth medium to 20,000 cells/ml. Transfer 100 ~1 of cells to each well in a 96-well flat bottom plate to give 2000 cells/well.
3. Allow half of plate (4 rows) for each compound and quadruplicate wells for each compound concentration, ' 2192797 a set of 4 wells far medium control and 4 wells for DMSO
control.
4. Gently shake plates to allow for uniform attachment of the cells to the plate.
5. Incubate the plates at 37°C in a 10% C02 incubator.
Day 1: Addition of Compound: same as for Assay 1.
Day 5: Devel~~pment of Assay: same as for Assay 1.
6.2.3. 3T3 Cell Growth Assay Assay 1: PDG:E'-Induced BrdU Incorporation Assay Materials and Reagents:
(1) PDG:E: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany (2) BrdU Labeling Reagent: 10 mM, in PBS
(pH'7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(3) Fixl~enat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(5) TMB Substrate Solution:
tetramethylbenzidine (TMB), ready to use, Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(6) PBS Washing Solution . 1X PBS, pH 7.4, made 3 0 in house .
(7) Albumin, Bovine (BSA): fraction V powder; A-8557_, Sigma Chemical Co. , USA.
Protocol (1) 3T3 engineered cell line: 3T3/EGFRc7.

(2) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2mM Gln in a 96 well plate. Cells are incubated overnight at 37C in 5% C02.

(3) ' After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24.hours.

(4) On day 3, ligand (PDGF=3.8 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM

with 0.1% BSA only; the positive control cells receive the ligand (PDGF) but no test compound:

Test compounds are prepared in serum free DMEM

with ligand in a 96 well plate, and serially diluted for ? test concentrations.

(5) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 ~M) for 1.5 hours.

(6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat~M

solution is added (50 ~1/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

(7) The FixDenat~solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated~milk in PBS, 200 ~ljwell) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.

(8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%

BSA) is added (100 ~1/well) and the plate is incubated.for 90 minutes at room temperature on a plate shaker.
(9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.
(10) TMB substrate solution is added (100 ~1/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
(11) The absorbance of the samples are measured at 410 nm (in "dual wavelength" mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech~~LISA plate reader.
Assay 2: EGF-Induced BrdU Incorgoration Assay Materials and Reagents (1) EGF: mouse EGF, 201; Toyobo,Co., Ltd. Japan (2) BrdU~Labeling Reagent: 10 mM, in PBS
(pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat~ fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(5) TMB Substrate Solution:
_ tetramethylbenzidine (TMB), ready to use, Cat.
No..i 647 229, Boehringer Mannheim, Germany.
(6) PBS Washing Solution : 1X PBS, pH 7.4, made in house.
(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co.,~USA.
_ 88 -Protocol (1) 3T3 engineered cell line: 3T3/EGFRc7 (2) Cells are seeded at 8000 cells/well in 10% CS, 2mM Gln in DMEM, in a 96 well plate.

Cells are incubated overnight at 37C in 5% C02.

(3) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(4) On day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM

with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound.

Test compounds are prepared in serum free DMEM

with ligand in a 96 well plate, and serially diluted for 7 test concentrations.

5) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 ~aM) for 1.5 hours.

6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 ~l/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

(7) The FixDenat~solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 ~C1/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.

(8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%

_ 89 _ BSA) is added (100 ;C1/well) and the plate is incubated for 90 minutes at room temperature on a.plate shaker.
(9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.
(10) TMB substrate solution is added (100 ~1/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
(11) The absorbance of the samples are measured at 410 nm (in "dual wavelength" mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech~~LISA plate reader.
Assay 3: EGF-Induced Her2 -Driven BrdU Incorporation Materials and Reaaents:
(1) EGF: mouse EGF, 201; Toyobo,Co., Ltd. Japan (2) BrdU Labeling Reagent: 1O mM, in PBS
(pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat~ fixation solution (ready to use), Cat. No. l 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(5) TMB Substrate Solution.
tetramethylbenzidine (TMB), ready to use, Cat.
No. l 647 229, Boehringer Mannheim, Germany.
(6) PBS Washing Solution : 1X PBS, pH 7.4, made in house.
(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.

Protocol:

(1) 3T3 engineered cell line:

3T3/EGFr/Her2/EGFr (EGFr with a Her2 kinase doma in (2) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2mM Gln in a 96- well plate.

Cells are incubated overnight at 37- in 5% COZ.

(3) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(4) On day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM

with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound.

.Test compounds are prepared in serum free DMEM

with ligand in a 96 well plate, and serially diluted for 7 test concentrations.

5) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 ~M) for 1.5 hours.

(6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 ~,1/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

(7) The FixDenat~solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 ~al/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.

(8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%

BSA) is added (100 ~1/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.

(9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.

(10) TMB substrate solution is added (100 ~cl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.

(11) The absorbance of the samples are measured at 410 nm (in "dual wavelength" mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatec~~ELISA plate reader.

Assay 4: ~aFi-Induced Erdv Incorporation Assay Materials and Reagents:
(1) IGF1 Ligand: human, recombinant; 6511, Promega Corp, USA.
(2) BrdU Labeling Reagent: 10 mM, in PBS
(pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat:l fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(5) TMB Substrate Solution:
tetramethylbenzidine (TMB), ready to use, Cat.
No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution : 1X PBS, pH 7.4, made in house.
(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.
' 5 Protocol:

(1) 3T3~engineered cell line: 3T3/IGFlr.

(2) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2mM Gln in a'96- well plate.

Cells are incubated overnight at 37C in 5% COZ.

(3) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(4) On day 3, ligand (IGF1=3.3 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM

with 0.1% BSA only; the positive control cells receive the ligand (IGF1) but no test compound.

Test compounds are prepared in serum free DMEM

with ligand in a 96 well plate, and serially diluted for 7 test concentrations.

5) After 16 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated .with BrdU (final concentration=10 ACM) for 1.5 hours.

(6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat~

solution is added (50 ~1/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

(7) The FixDena~~solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 ~clJwell) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.

(8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%

BSA) is added (100 ~cl/well) and the plate is incubated.for 90 minutes at room temperature on a plate shaker.

(9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.

(10) TMB substrate solution is added (100 ~C1/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.

(1l) The absorbance of the samples are measured at 410 nm (in "dual wavelength" mode with a filter 2o reading at 490 nm, as a reference wavelength) on a Dynatechl ELISA.plate reader.

Assay 5: Insulin-Induced BrdU Incorporation Assay Materials and Reagents:
(1) Tnsulin: crystalline, bovine, Zinc; 13007, Gibco BRL, USA.
(2) BrdU Labeling Reagent: 10 mM, in PBS
(pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat~s fixation solution (ready to use), .
Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat: No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution:
tetramethylbenzidine (TMB), ready to use, Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(6) PBS Washing Solution : 1X PBS, pH 7.4, made in house.
(7) Albumin, Bovine (BSA): fraction V powder,~ A-8551, Sigma Chemical Co., USA.
Protocol:

(1) 3T3 engineered cell line: H25 (2) Cells are seeded at 8000 cellsjwell in DMEM, 10% CS, 2mM Gln in a 96 well plate. Cells are incubated overnight at 37C in 5% C02.

(3) After 24 hours, the cells are washed with i5 PBS, and then are serum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(4) On day 3, ligand (Insulin=10 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the.cells simultaneously. The negative control wells receive serum free DMEM

with 0.1% BSA only; the positive control cells receive the ligand (Insulin) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.

(5) After 16 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells~are incubated with BrdU (final concentration=10 ACM) for 1.5 hours.

(6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat~i solution is added (50 ~Cljwell) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

(7) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 ~C1/well) as a blocking solution and , the plate is incubated for 30 minutes at room temperature on a plate shaker.

(8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%

14 BSA) is added . (100 ~1/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.

(9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.

(10) TMB substrate solution is added (100 ~Sl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.

(11) The absorbance of the samples are measured at 410 nm (in "dual wavelength" mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.

6.2..4. HOV-EC-C Assay The following protocol may also be used to measure a compound's activity:

1. Wash_and trypsinize HUV-EC-C cells (human umbilical vein endothelial cells, (American Type Culture Collection; catalogue no. 1730 CRL). Wash with Dulbecco~s~phosphate-buffered saline (D-PBS; obtained from Gibco BRL; catalogue no. 14190-029) 2 times at about 1 m1/10 cm2 of tissue culture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic cell dissociation solution (Sigma Chemical Company; catalogue no. C-1544).
The 0.05% trypsin was made by diluting 0.25% trypsin/1 mM
EDTA (Gibco; catalogue no. 25200-049) in the cell dissociation solution. Trypsinize with about 1 m1/25-30 cm2 of tissue culture flask for about 5 minutes at 37°C.
After cells have detached from the flask, add an equal volume of assay medium and transfer to a 50 ml sterile centrifuge tube (Fisher Scientific; catalogue no. 05-539-6) .
l0 2. Wash the cells with about 35 ml assay medium in the 50 ml sterile centrifuge tube by adding the assay medium, centrifuge for to minutes at approximately 200xg, aspirate the supernatant, and resuspend with 35 ml 'D-PBS.
Repeat the wash two more times with D-PBS, resuspend the cells in about 1 ml assay medium/15 cmz of tissue culture flask. Assay medium consists of F12K medium (Gibco BRL;
catalogue no. 21127-014) + 0.5% heat-inactivated fetal bovine serum. Count the cells with a Coulter Counter~v CoulterTM Electronics, Inc.) and add assay medium to the cells to obtain a concentration of 0.8-1.0x105 cells/ml.
3. Add cells to 96-well flat-bottom plates at 100 ~,1/well or 0.8-1.0x104 cells/well; incubate ~24h at 37°C, 5% C02.

1. Make up two-fold drug titrations in separate 96-well plates, generally 50 ~cM on down to 0 ;cM. Use the same assay~medium as mentioned in day 0, step 2 above.
Titrations are made by adding 90 ~1/well of drug at 200 ~tM (4X the final well concentration) to the top well of a particular plate column. Since the stock drug concentration is usually 20 mM in DMSO, the 200 ~,M drug concentration contains 2$ DMSO.
Therefore, diluent made up to 2% DMSO in assay medium (F12K + 0.5% fetal bovine serum) is used as diluent for the drug titrations in order to dilute the drug but keep the DMSO concentration constant. Add this diluel~t to the remaining wells in the column at 60 ~,1/well. Takes 60 ~,1 from the 120 ~,l of 200 ~M drug dilution in the top well of the column and mix with the 60 ~,1 in the :second well of the column. Take 60 ~,1 from this well and mix with the 60 ~,1 in the third well of the column, and so on until two-fold titrations are completed. When the next-to-the-last well is mixed, take 60 ~,1 of the :L20 ~cl in this well and discard it. Leave the last well with 60 ~,1 of DMSO/media diluent as a non-drug-containing control. Make 9 columns of titrated drug, enough i:or triplicate wells each for 1) VEGF
(obtained from Pepro Tech Inc., catalogue no. 100-200, 2) endothelial cell growth factor (ECGF) (also known as acidic fibrob~Last growth factor, or aFGF) (obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600), and assay med~.a control. ECGF comes as a preparation with sodium hE;parin.
2. Transfer 50 ~l/well of the drug dilutions to the 96-well a.>say plates containing the 0.8-1.0x104 cells/100 ~,l/well of the HUV-EC-C cells from day 0 and 2 0 incubate --2 h at 3 7"C , 5 % COZ .
3. In triplicate, add 50 ~,1/well of 80 ug/ml VEGF, ng/ml ECGF, or media control to each drug condition.
As with the drugs, the growth factor concentrations are 4X the desired final concentration. Use the assay media from day 0 step 2 to make the concentrations of growth factors. Incubate approximately 24 hours at 37°C, 5% COz.
Each well will_ have 50 ~1 drug dilution, 50 ~,1 growth factor or media, and 100 u1 cells, - 200 ul/well total.
Thus the 4X concentrations of drugs and growth factors become 1X once everything has been added to the wells.

1. Add 3H-thymidine (Amersham; catalogue no. TRK-686) at 1 ~.Ci/'well_ (10 ~.l/well of 100 ~,Ci/ml solution made up in RPriI media + 10% heat-inactivated fetal bovine serum) and incubate -24 h at 37°C, 5% CO2. Note: 3H-thymidine is made up in RPMI media because all of the other applications for which we use the 3H-thymidine involve experiments done in RPMI. The media difference at this step is probably not significant. RPMI was obtained from Gibco BRL, catalogue no. 11875-051.

1. Freeze plates overnight at -20°C.

1. Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester 96~) onto filter mats (Wallac; catalogue no. 1205-401); read counts on a Wallac 1o Betaplate~T°''~ liquid scintillation counter.
6.2.5. PDGF-R Cellular Assay The PDGF cellular kinase assay was carried out as follows: cells are lysed in 0.2 M Hepes, 0.15 M NaCl, 10$
V/V glycerol, 0.04% Triton X-100, 5 mM EDTA, 5 mM sodium vanadate and 2 mM Na+ pyrophosphate; cell lysates are then added to an ELISA plate coated with an anti-PDGF
receptor antibody (Genzyme); ELISA plates are coated at 0.5 ~g of antibody/well in 150 ~1 of PBS for 18 hours at 4°C prior to the addition of the lysate; the lysate is incubated in the coated plates for d hour and then washed four times in TBST (35 mM Tris-HC1 pH 7.0, 0.15 M NaCl, 0.1% Triton X100); anti-phosphotyrosine antibody (100 ~tl in PBS) is added and the mixture. is incubated for 30 minutes at room temperature; the wells were then washed four times in TBST, a secondary antibody conjugated to POD (TAGO) is added to each well, and the treated well are incubated for 30 minutes at room temperature; the wells are then washed four times in TBST, ABTS/Hz02 solution is added to each well and the wells are incubated for two minutes; absorbance is then measured at 410 nm.

~192T97 6.2..6. Experimental Results Of Cell Growth Assay Results for various compounds obtained from the abovEa-described assays are set forth in the Tables that follow:

Nfitogenesis in Endothelial Cells (3H~Thymidine Incorporation COMF~OUND HW-EC Assay (Example) VEGF (~,M) a-FGF (ACM) 27 1.1 153.8 1B 0.2 6.0 16 6.6 3.4 17 4.8 35.7 479f~ 30.7 35.8 20 43.2 21 19.9 22 2.5 45.2 23 1.6 4.6 24 14.8 25 3.4 3.7 26 25.6 19.3 28 8.0 13.0 29 34.3 16.3 30 1.0 1.4 32 4.4 4.9 4 0.6 33 46.1 27.3 5 0.8 25.8 5201. 2.5 2.3 36 2.3 0.7 37 5.1 11.8 38 2.9 130 5217 9.6 10.5 2 i ~~27~7 COMF~OUND HW-EC Assay (Exa.mple) VEGF (~,M) a-FGF (~M) 41 2.4 2.7 8 2.2 42 <0.8 2.0 9 <0.8 31.1 44 0.9 0.6 10 <0.8 45 39.8 35.5 11 <0.8 22.7 5409' 26.0 12 <0.8 48 13.6 40 13 0.7 50 11.4 14 2.5 15 5.7 5429' 27.6 59 0.16 0.14 60 39.8 33.0 61 1.2 30.0 63 3.8 3.4 68 <0.07 <0.07 69 0.5 0.8 70 0.14 7.9 71 3.8 12.9 73 1.3 3.2 74 0.54 8.7 76 2.0 5.0 77 1.2 14.1 81 0.05 37.8 COMI?OUND HUV-EC Assay (Example) VEGF (~,M) a-FGF (~M) 7 1.2 3.8 Mitogenesis in 3T3/EGFR Cells BrdU Incorporation COMPOUND PDGFR FGFR EGFR
PDGF Ligand FGF Ligand EGF Ligand (Example) IC50 (uM) IC50 (~eM) IC50 (~M) 4313 6 5.5 5.5 1B 2.5 29 9 4.9 60 10 5.2 45 7.5 70 100 12 2.8 70 74 g 81 6.5 9 48 ' 2 ~ 92791 Cell Growth Assay on Various Cell Lines SRB Readout COMPOUND :3T3/E/H+ 3T3/EGFR+ 3T3/PDGFR+ SMC
9~GF-a(T) TGF-a(T) PDGF(T) (Example) IC50 (~,M) IC50 (~,M) IC50 (ACM) IC50 (~,M) 4313 32 10.7 8.8 105 22.2 3T3/E/H+'rGF-cz(T): NIH 3T3 cells expressing EGFR/HER2 chimera and TGF-a, tumor-derived 3T3/EGFR-+-TGF-a(T): NIH 3T3 cells expressing EGFR and TGF-a, tumor-<3erived 3T3/PDGFI~+PDGF(T): NIH 3T3 cells expressing PDGF-(3R
and PDGF-~3~3, -tumor-derived SMC: human :smooth muscle cells from Clonetics 6.3. Measurement Of Cell Toxicity Therapeui~ic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cyi~otoxic effect. A measure of the effectiveness and cell toxicity of a compound can be obtained by dc=termining the therapeutic index: ICso/LDS~-ICSO, the dose required to achieve 50% inhibition, can be measured usin<~ standard techniques such as those described herein. LDso,the dosage which results in 50%
toxicity, can also be measured by standard techniques (Mossman, 1983, J- Immunol. Methods, 65:55-63), by measuring the amount of LDH released (Korzeniewski and Callewaert, 1~~83, J. Immunol. Methods 64:313; Decker and Lohmann-MatthE~s, 1988, J. Immunol. Methods 115:61), or by measuring the lethal dose in animal models. Compounds with a large i:herapeutic index are preferred. The therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50.

?92197 6.3. In vivo Animal Models 6.3.1. Xenograft Animal Models The ability of human tumors to grow as xenografts in athymic mice (e. g., Balb/c, nu/nu) provides a usE~ful in vivo model for studying the biological re:~ponse to therapies for human tumors. Since the first successful xenotransplantation of human tumors into athymic mice, (Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760), many different human tumor csall lines (e. g., mammary, lung, genitourinary,, gastrointestinal, head and neck, glioblastoma, bone, and malignant melanomas) have been transplanted and successfully grown in nude mice. Human mammary tumor cell lines, including MCF-7, ZR75-l, and MDA-MB-231, hive been established as subcutaneous xenografts in nude mice (Warri et al., 1991, Int. J.
Cancer 49:616--623; Ozzello and Sordat, 1980, Eur. J.
Cancer 16:553--559; Osborne et al., 1985, Cancer Res.
45:584-590; Seibert et al., 1983, Cancer Res. 43:2223-2239) .
Assay 1: HER~?/Xenograft Animal Model To study the effect of anti-tumor drug candidates on HER2 expressing tumors, the tumor cells should be able to grow in the absence of supplemental estrogen. Many mammary cell 7_ines are dependent on estrogen for in vivo growth in nudE~ mice (Osborne et al., supra), however, exogenous estrogen suppresses HER2 expression in nude mice (Warri et: al., supra, Dati et al., 1990, Oncogene 5:1001-1006). For example, in the presence of estrogen, MCF-7, ZR-75-J_, and T47D cells grow well in vivo, but express very l.ow levels of HER2 (Warri et al., supra, Dati et al., ~cupra).
The following type of xenograft protocol can be used:
1) implant tumor cells (subcutaneously) into the hindflank of five- to six-week-old female Balb/c nu/nu athymic mice;

' 2?92197 2) administer the anti-tumor compound;
3) mean>ure tumor growth by measuring tumor volume.
The tumors can also be analyzed for the presence of a receptor, such as HER2, EGF or PDGF, by Western and immunohistochE:mical analyses. Using techniques known in the art, one ~;kill.ed in the art can vary the above procedures, for example through the use of different treatment regimes.
Assay 2: FLK-1/Xenograft Model.
The ability of the compounds of the present invention to inhibit ovarian, melanoma, prostate, lung and mammary tumor cell lines established as SC xenografts was examined. These studies were conducted using doses ranging from 1. to 75 mg/kg/day.
Materials And Methods. The tumor cells were implanted subc:utaneously into the indicated strains of mice. Treatment was initiated on day 1 post implantation unless otherwise indicated (e. g. treatment of the SCID
mouse related to the A375 melanoma cell line began on Day 9). Eight (8) to sixteen (16) mice comprised each test group.
Specif ica.lly:
Animals. Female athymic mice (BALB/c, nu/nu), BALB/c mice, Y;iistar rats and Fisher 344 rats were obtained from Simonsen Laboratories (Gilroy, CA). Female A/I mice were obtained from Jackson Laboratory (Bar Harbor, ME). DA rats were obtained from B&K Universal, Inc. (Fremont, CA). Athymic R/Nu rats, DBA/2N mice, and BALB/c mice were obtained from Harlan Sprague Dawley (Indianapolis, IN). Female C57BL/6 mice were obtained from Taconic (Germantown, NY). All animals were maintained under clean-room conditions in Micro-isolator cages with Al~~ha-dri bedding. They received sterile rodent chow ar.~d water ad libitum.
All procedures were conducted in accordance with the NIH Guide for the Care and Use Of Laboratory Animals.

Subcutaneous Xenograft Model. Cell lines were grown in appropriate medium as described (See Section 6).
Cells were harvested at or near confluency with 0.05%
Trypsin-EDTA and pelleted at 450 x g'for 10 min. Pellets were resuspended in sterile PBS or media (without~FBS) to a suitable concentration indicated in the Figure legends and the cells were implanted into the hindflank of mice.
Tumor-growth was measured over 3 to 6 weeks using venier calipers and tumor volumes were calculated as a product of length x width x height unless otherwise indicated. P
values were calculated using the Students' t-test.
Compound in 50 - 100 ~L excipient (dimethylsulfoxide, PETE, PBTE6C:D5W, or PHTE:DSW) was delivered by IP
injection at concentrations indicated in the Figure v legends.
Intracerebral Xenograft Model. For the mouse IC model, rat C6 glioma cells were harvested and suspended in sterile PHS at a concentration of 2.5 x 10' cells/ml and placed on ice. Cells were implanted into BALB/c, nu/nu mice in the following manner: the frontoparietal scalps of mice were shaved with animal clippers if necessary before swabbing with 70% ethanol.
Animals were anesthetized with isofluorane and the needle was inserted through the skull into the left hemisphere of the brain. Cells were dispensed from Hamilton Gas-tight Syringes using 30 ga 1/2 inch needles 'fitted with sleeves that allowed only a 3 mm penetration. A repeater dispenser was used for accurate delivery of 4 ~uL of cell suspension. Animals were monitored daily for well-being and were sacrificed when they had a weight loss of about - 40% and/or showed neurological symptoms.
For the rat IC model, rats (Wistar, Sprague Dawley, Fisher 344, or athymic R/Nu; approximately 200-400 g (some 3-400g)) were anesthetized by an IP injection of 100 mg/kg Ketaset~(ketamine hydrochloride; Aveco, Fort Dodge, Iowa) and 5 mg/kg Rompur~M(xylazine, 2% solution;
Bayer, Germany). After onset of anesthesia, the scalp was shaved and the animal was oriented in a stereotaxic apparatus (Stoelting, Wood Dale, IL). The skin at the incision site was cleaned 3. times with alternating swabs of 70% ethanol and.l0% Povidone-Iodine . A median l:0 -1.5 cm incision was made in the scalp using a~sterile surgical blade. The skin was detached slightly and pulled to the, sides to expose the sutures on the skull surface. A dental drill (Stoelting, Wood Dale, IL) was used,to make a small (1-2 mm diameter) burrhole in the skull approximately 1 mm anterior and 2 mm lateral to the bregma. The cell suspension was drawn into a 50 ~,L
. Hamilton syringe fitted with a 23 or 25g standard bevel needle. The syriwge was oriented in the burrhole at the level of the arachnoidea and lowered until the tip of the needle was 3 mm deep:into the brain structure,~where the cell suspension was slowly injected. After cells were injected, the needle was left in the burrhole for 1-2 .
minutes to allow for.complete delivery of the cells. The skull was cleaned and the skin was closed with 2 to 3 sutures. Animals were observed 'for recovery from surgery and anesthesia. Throughout the~experiment, animals were observed at least twice each day for development of 'symptoms associated with progression of intracerebral tumor. Animals displaying advanced symptoms (leaning, 25.loss of balance, dehydration, loss of appetite, loss of coordination, cessation of grooming activities, and/or significant weight.loss) were humanely sacrificed and the organs and tissues of interest were resected.
Tntraperitoneal Model. Cell lines were grown in the appropriate media. Cells were harvested' and washed in sterile PBS or medium without FBS, resuspended to .a suitable concentration, and injected into_the IP
cavity of mice of the appropriate strain. Mice were observed daily for the occurrence of ascites formation:
Individual animals were sacrificed when they presented with a weight gain of 40%, or when the IP tumor burden' began to cause undue stress and pain to the animal.

~ 192797 6.3.2. In Vlvo VEGF Pellet Model In the following example, the Pellet Model was used to test a compound's activity against the FLK-1 receptor and against disorders associated with the formation of blood vessels. In this model, VEGF is packaged into a time-release pellet and implanted subcutaneously on the abdomen of nude mice to induce a 'reddening' response and subsequent swelling around the pellet. Potential FLK-1 inhibitors may then be implanted in methylcellulose near the VEGF pellet to determine whether such inhibitor may be used to inhibit the "reddening" response and subsequent swelling.
Materials And Methods. The following materials were used:
1) VEGF- human recombinant lyophilized product is commercially available and may be obtained from Peprotech, Inc., Princeto~z Business Park, G2; P.O. box 275, Rocky Hill, NJ 08553.
2) VEGF packaged into 21 day release pellets were obtained from ~Cnnovative Research of America (Innovative Research of Ame=rica, 3361 Executive Parkway, P.O. Box 2746, Toledo, Ohio 43606), using patented matrix driven delivery system. Pellets were packaged at 0.20, 0.21, or 2.1 ~Cg VEGF/pel.let. These doses approximate 10 and 100 ng/day release of VEGF.
3) Methylcellulose 4) Water (sterile) 5) Methanol 6) Appropriate drugs/inhibitors 7) 10 cm; culture plates 8) parafilm The following protocol was then followed to conduct the VEGF pellet model:
1) VEGF, purchased from Peprotech, was sent to Innovative Research for Custom Pellet preparation;
2) Methylcellulose prepared at 1.5% (w/v) in sterile water;

A

~i92T97 3) Drugs solubilized in methanol (usual concentration range = 10 to 20 mg/ml);
4) Place sterile parafilm in sterile 10 cm plates;
5) 150 ~,1 of drug in methanol added to 1.35 ml of 1.5o methylcellulose and mixed/vortexed thoroughly;
6) 25 ~cl aliquots of homogenate placed on parafilm and dried into discs;
7) Mice (6-10 wk. Balb/C athymic nu/nu, female) were anesthetized via isoflurane inhalation; 8) VEGF pellets and methylcellulose discs were implanted subcutaneously on the abdomen; and 9) Mice were scored at 24 hours and 48 hours for reddening and swelling response.
The specific experimental design used in this example was:
N = 4 animals/group Controls: VEGF pellet + drug placebo VEGF placebo + drug pellet Experime~ztal Results. The compounds of the present invention are expected to demonstrate activity according to this assay.
6.3.3. Mammary Fat Pad Model Because of the established role played by mane of the RTKs, e.g., the HER2 receptor, in breast cancer,, the mammary fat pad model is particularly useful for measuring the efficacy of compounds which inhibit such RTKs. By implanting tumor cells directly into the locai:ion of interest, in situ models more accurately rei=lect the biology of tumor development than do subcutaneous models. Human mammary cell lines, including MCF--7, have been grown in the mammary fat pad of athymic mice. Shafie and Grantham, 1981, Natl. Cancer Instit. 67:51--56; Gottardis et al., 1988, J. Steroid Biochem. 30:3~~1-314. More specifically, the following procedure can be used to measure the inhibitory effect of a compound on the HER2 receptor:

- 2192i'97 1) Impl;~nt, at various concentrations, MDA-MB-231 and 1KCF-7 cells transfected with HER-2 into the axil:lary mammary fat pads of female athymic mice;
2) Administer the compound; and 3) Measure the tumor growth at various time points .
The tumora can also be analyzed for the presence of a receptor such as HER2, by Western and immunohistochemical analyses. Using techniques known in the art, one spilled in the art can vary the above procedures, four example through the use of different treatment regimes.
6.3.4. Tumor Invasion Model The :Following tumor invasion model has been developed and may be used for the evaluation of therapeutic va:Lue and efficacy of the compounds identified to :selectively inhibit KDR/FLK-1 receptor.
6.3.4.1. Procedure 8 week old nude mice (female) (Simonsen Inc.) were used as experimental animals. Implantation of tumor cells waa performed in a laminar flow hood. For anesthesia, Xy:lazine/Ketamine Cocktail (100 mg/kg ketamine and 5 mg/kg) are administered intraperitoneally.
A midline inci:~ion is done to expose the abdominal cavity (approximately 1.5 cm in length) to inject 10' tumor cells in a volume of 100 ~C1 medium. The cells are injected either into thca duodenal lobe of the pancreas or under the serosa of i~he colon. The peritoneum and muscles are closed with a ~~-0 silk continuous suture and the skin was closed by using would clips. Animals were observed daily.
6.3.4.2. Analysis 1 '~~797 After 2-6 weeks, depending on gross observations of the animals, the mice are sacrificed, and the local tumor metastases, to various organs (lung, liver, brain, stomach, spleen, heart, muscle) are excised and analyzed (measurements of tumor size, grade of invasion, immunochemistry, and in situ hybridization).
6.3.5. RESULTS
Results for various compounds obtained from the above-described in vivo assays are set forth at Table 5, below:

In v~vo Data COI~OUND EpH4-VEGF

(Example) %inhibition @ mg/kg .

27 56% @ ?5 ~
_ _ .
' . _ __ 50% @ 75 63% @ 50 22 42% @ 75 __--___ __ 42% @ 50/50 4942 46% @ 50 4?% @ 25 12 50% @ 25 ______ , ___ _____~___ 57% @ 3?.5/37.5 14 45% @ 50 65% @ 50 15 47% @ 50 65% @ 50 The present invention is not to. be limited in scope bY ~e exemplified embodiments which are intended as illustrations of single aspects of the invention, and any clones, DNA or amino acid sequences which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims (15)

We claim:
1. A compound of the formula:
and pharmaceutically acceptable salts thereof, wherein:
R1 is H;
R2 is O or S;
R3 is hydrogen;
R4, R5, R6, and R7 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO2NRR', SO3R, SR, NO2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH2)n CO2R, and CONRR';
A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, and tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO2NRR', SO3R, SR, NO2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH2)n CO2R, or CONRR';
n is 0-3;
R is H, alkyl or aryl; and R' is H, alkyl or aryl;
wherein in the above definitions:
alkyl refers to a straight chain, branched or cyclic saturated aliphatic hydrocarbon having 1 to 12 carbon atoms and optionally being substituted with one or more substituents selected from hydroxy, cyano, =O, =S, NO2, halogen, N(CH3)2, amino and -SH;
aryl refers to aromatic group which has at least one ring having a conjugated pi electron system including carbocyclic aryl, heterocyclic aryl and biaryl groups and optionally being substituted with one or more substituents selected from the halogen, trihalomethyl, hydroxyl, SH, OH, NO2, amine, thioether, cyano, alkoxy, alkyl, and amino;
alkaryl refers to an alkyl that is covalently bound to an aryl group; and alkoxy, aryloxy and alkaryloxy refer to a -O-alkyl, -O-aryl and -O-alkaryl group, respectively;
with the proviso that the following compounds are excluded:
3-(pyrrol-2-ylmethylene)-2-indolinone;
3-(5-chloro-3,4-dimethylpyrrol-2-ylmethylene)-2-indolinone ;
3-(3,5-dimethyl-4-ethylpyrrol-2-yl)-2-indolinone;
3-(3,5-dimethyl-4-ethoxycarbonylpyrrol-2-yl)-2-indolinone;
3-[(1-methyl-5-nitro-imidazol-2-yl)methylene]-2-indolinone;
3-(thien-2-ylmethylene)-2-indolinone;

1H-indole-7-acetic acid, 3-[(2-butyl-1H-imidazol-4-yl)methylene]-2,3-dihydro-2-oxo-, ethyl ester;
1H-indole-7-acetic acid, 3-[[2-butyl-1-[(1,1-dimethylethoxy)carbonyl]-1H-imidazol-4-yl]methylene]-2,3-dihydro-2-oxo-, ethyl ester;
5-benzoyl-3-[(imidazole-2-yl)methylene]-2-indolinone;
6-diethylamino-3-[(isothiazole-2-yl)methylene]-2-indolinone;
5-chloro-3-[(thiazole-2-yl)methylene]-2-indolinone; and 6-nitro-3-[(pyrrole-2-yl)methylene]-2-indolinone.
2. A compound of the formula:
and pharmaceutically acceptable salts thereof, wherein:
R1 is H;
R2 is O or S;
R3 is hydrogen;
R4, R5, R6, and R7 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO2NRR', SO3R, SR, NO2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH2)n CO2R, and CONRR';

A is a five membered heteroaryl ring selected from the group consisting of pyrazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, and tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO2NRR', SO3R, SR, NO2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH2)n CO2R, or CONRR';
n is 0-3;
R is H, alkyl or aryl; and R' is H, alkyl or aryl;
wherein, in the above definitions, alkyl, aryl, alkaryl, alkoxy, aryloxy and alkaryloxy have the definitions as given in claim 1;
with the proviso that the following compounds are excluded:
6-diethylamino-3-[(isothiazole-2-yl)methylene]-2-indolinone; and 5-chloro-3-[(thiazole-2-yl)methylene]-2-indolinone.
3. A compound according to claim 1 selected from the group consisting of:
3-[(3-methylpyrrol-2-yl)methylene]-2-indolinone;
3-[(3,4-dimethylpyrrol-2-yl)methylene]-2-indolinone;
3-[(2-methylthien-5-yl)methylene]-2-indolinone;
3-[(3-methylthien-2-y1)methylene]-2-indolinone;
3-{[4-(2-methoxycarbonylethyl)-3-methylpyrrol-5-yl]methylene}-2-indolinone;
3-[(4,5-dimethyl-3-ethylpyrrol-2-yl)methylene]-2-indolinone;

3-[(5-methylimidazol-2-yl)methylene]-2-indolinone;
5-chloro-3-[(5-methylthien-2-yl)methylene]-2-indolinone;
3-[(3,5-dimethylpyrrol-2-yl)methylene]-5-nitro-2-indolinone;
3-[(3-(2-carboxyethyl)-4-methylpyrrol-5-yl)methylene]-2-indolinone;
5-chloro-3-[(3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone; and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone;
or a pharmaceutically acceptable salt thereof.
4. The compound of Claim 3, wherein said compound is 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone, or a pharmaceutically acceptable salt thereof.
5. A pharmaceutical composition comprising a compound of any of claims 1 to 4 or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or excipient.
6. A pharmaceutical composition according to claim 6 wherein said pharmaceutical composition is suitable for parenteral or subcutaneous administration or is in a depot formulation.
7. Use of a compound for the manufacture of a pharmaceutical composition for treating diseases related to unregulated tyrosine kinase signal transduction, said compound having the formula:

and pharmaceutically acceptable salts thereof, wherein:
R1 is H or alkyl;
R2 is O or S;
R3 is hydrogen;
R4, R5, R6, and R7 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO2NRR', SO3R, SR, NO2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH2)n CO2R, and CONRR';
A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, and tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO2NRR', SO3R, SR, NO2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH2)n CO2R, or CONRR';
n is 0-3;
R is H, alkyl or aryl; and R' is H, alkyl or aryl;
wherein, in the above definitions, alkyl, aryl, alkaryl, alkoxy, aryloxy and alkaryloxy have the definitions as given in claim 1.
8. Use of a compound for the manufacture of a pharmaceutical composition for regulating, modulating or inhibiting tyrosine kinase signal transduction, said compound having the formula:
and pharmaceutically acceptable salts thereof, wherein:
R1 is H or alkyl;
R2 is O or S;
R3 is hydrogen;
R4, R5, R6, and R7 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO2NRR', SO3R, SR, NO2, NRR', OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH2)n CO2R, and CONRR';
A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, and tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO2NRR' , SO3R, SR, NO2, NRR' , OH, CN, C (O) R, OC (O) R, NHC (O) R, (CH2) nCO2R, or CONRR' ;

n is 0-3;

R is H, alkyl or aryl; and R' is H, alkyl or aryl;

wherein, in the above definitions, alkyl, aryl, alkaryl, alkoxy, aryloxy and alkaryloxy have the definitions as given in claim 1.
9. The use of claim 7 wherein said disease is selected from the group consisting of cancer, blood vessel proliferative disorders, fibrotic disorders, mesangial cell proliferative disorders and metabolic diseases.
10. The use of claim 9 wherein the blood vessel proliferative disorder is selected from the group consisting of arthritis and restenosis.
11. The use of claim 9 wherein the fibrotic disorder is selected from the group consisting of hepatic cirrhosis and atherosclerosis.
12. The use of claim 9 wherein the mesangial cell proliferative disorder is selected from the group consisting of glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes;
transplant rejection and glomerulopathies.
13. The use of claim 9 wherein the metabolic disease is selected from the group consisting of psoriasis, diabetes mellitus, wound healing, inflammation and neurodegenerative diseases.
14. The use of claim 7 or 8 wherein the compound is selected from the group consisting of:

3-[(3-methylpyrrol-2-yl)methylene]-2-indolinone;
3-[(3,4-dimethylpyrrol-2-yl)methylene]-2-indolinone;
3-[(2-methylthien-5-yl)methylene]-2-indolinone;
3-[(3-methylthien-2-yl)methylene]-2-indolinone;
3-{[4-(2-methoxycarbonylethyl)-3-methylpyrrol-5-yl]methylene}-2-indolinone;
3-[(4,5-dimethyl-3-ethylpyrrol-2-yl)methylene]-2-indolinone;
3-[(5-methylimidazol-2-yl)methylene]-2-indolinone;
5-chloro-3-[(5-methylthien-2-yl)methylene]-2-indolinone;
3-[(3,5-dimethylpyrrol-2-yl)methylene]-5-nitro-2-indolinone;
3-[(3-(2-carboxyethyl)-4-methylpyrrol-5-yl)methylene]-2-indolinone;
5-chloro-3-[(3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone; and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone;
or a pharmaceutically acceptable salt thereof.
15. The use of claim 14 wherein the compound is 3-[(2,4-dimethylpyrrol-5-yl)methylene)-2-indolinone or a pharmaceutically acceptable salt thereof.
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